Citation
Phase II Biotransformation of Xenobiotics in Polar Bear (Ursus maritimus) and Channel Catfish (Ictalurus punctatus)

Material Information

Title:
Phase II Biotransformation of Xenobiotics in Polar Bear (Ursus maritimus) and Channel Catfish (Ictalurus punctatus)
Creator:
SACCO, JAMES C. ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Catfish ( jstor )
Chlorine ( jstor )
Complementary DNA ( jstor )
Enzymes ( jstor )
Intestines ( jstor )
Kinetics ( jstor )
Liver ( jstor )
Polar bears ( jstor )
Polymerase chain reaction ( jstor )
RNA ( jstor )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright James C. Sacco. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2006

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Full Text











PHASE II BIOTRANSFORMATION OF XENOBIOTICS IN POLAR BEAR
(Lh-sus maritimus) AND CHANNEL CATFISH (Ictalurus punctatus)














By

JAMES C. SACCO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

JAMES C. SACCO


































This document is dedicated to Denise and my parents.
















ACKNOWLEDGMENTS

First and foremost, I would like to thank my mentor, Dr. Margaret O. James, for

her instruction, guidance, and support throughout my PhD program. Through her

excellent scientific and mentoring skills I not only managed to complete several

interesting studies but also rekindled my scientific curiosity with regards to

biotransformation and biochemistry in general. I greatly appreciate the advice and

instruction of Ms. Laura Faux, our laboratory manager, on enzyme assays, HPLC, and

fish dissection. The assistance and advice of Dr. David S. Barber, Mr. Alex McNally,

and Mr. Jason Blum at the Center for Human and Environmental Toxicology in walking

me through the complexities of molecular cloning are much appreciated. Academic

discussions with Dr. Liquan Wang, Dr. Ken Sloan, Dr. Joe Griffitt and Dr. Nancy

Denslow also helped me to interpret my results and design better experiments

accordingly.

Last but not least, I would like to thank my fiancee, Denise, and my parents, for

their support and encouragement throughout my doctoral studies.




















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ................. ................. vii........ ....


LIST OF FIGURES .............. .................... ix


AB STRAC T ................ .............. xii


CHAPTER


1 BIOTRANSFORMATION AND ITS IMPORTANCE IN THE
DETOXIFICATION OF XENOBIOTICS ................. ...............1............ ....


2 PHASE II CONJUGATION: GLUCURONIDATION AND SULFONATION .........4


UJDP-Glucuronosyltransferases (UGTs) .....__.....___ ..........._ ............7
Sulfotransferases (SULTs) ............ ..... ._ ...............11....

3 SULFONATION OF XENOBIOTICS BY POLAR BEAR LIVER .........................14


Hypothesis .............. ...............17....
M ethodology ................. ...............17.................
Re sults ................ ...............23.................
Discussion ................. ...............32.................
Conclusions............... ..............3


4 GLUCURONIDATION OF POLYCHLORINATED BIPHENYLOLS BY
CHANNEL CATFISH LIVER AND INTESTINE ......____ ........_ ...............38


Hypothesis .............. ...............41....
M ethodology ................. ...............41....... .....
Re sults ....._ _................ ........_ _..........4
Discussion .........._ ......... ...... ...............53....
Conclusions and Recommendations .....__................. ........_ .........5


5 CLONING OF UDP-GLUCURONOSYLTRANSFERASES FROM CHANNEL
CATFISH LIVER AND INTESTINE............... ...............6


Piscine UGT Gene Structure and Isoforms ............. ..............60.....












Hypothesis ................ ...............62...
Method ology (part 1) ............... ...............62...
Results and discussion (part 1) .............. ...............74....
Methodology (part 2) .........__.. ..... ._ __ ...............79....
Overview of RLM-RACE ....._.. ................ ........_._ ......... 7
5' RLM-RACE procedure .............. ...............8 4....
3' RACE procedure ..................... ...............86.
PCR amplification of entire UGT gene ......___ ..... .._.. ......_._.........8
Results (part 2)............... ... ...............8
Nucleotide sequence analysis ................ ...............89........... ....
Protein sequence analysis ................ ...............100................
Cloning of entire UGT gene ................. ...............104........... ...
Discussion ................. ...............107................
Limitations ................. ............ ...............110......
Conclusions and recommendations ................. ......... ......... .............1


6 DETERMINATION OF PHYSIOLOGICAL UDPGA CONCENTRATIONS IN
CHANNEL CATFISH LIVER AND INTESTINE ......____ ........ __ ..............116


UDP-Glucuronic Acid (UDPGA) ................. ...............116................
Obj ective ................. ....._ __ ...............118......
M ethod Development ................. ...............118....... ......
Sample Digestion ....__ ................. .........__..........19
H PLC ............ _...... ...............1 1....
Final M ethod .............. ...............123....
Re sults............ ..... .. ...............125...
Discussion ............ __... ...._ ..... ._ ............12
Conclusions and Recommendations ....__ ......_____ .......___ .............2


APPENDIX


A SEQUENCES OF UGT PARTIAL CLONES AND AMPLICONS ................... .....131


B SEQUENCES FOR UGT FULL-LENGTH CLONES FROM CATFISH LIVER..138

LIST OF REFERENCES ........._._ ...... ..... ...............144....


BIOGRAPHICAL SKETCH ........._... ...... .___ ...............157....

















LIST OF TABLES


Table pg

2-1 Expression of human UGT mRNA in various tissues ................. ........... ..........9

2-2 Tissue distribution of SULTs (cDNA and mRNA) in humans.............. ...... ........._12

3-1 Estimated kinetic parameters (Mean + SD) for (a) sulfonation and (b)
glucuronidation of 3-OH-B[a]P by polar bear liver cytosol and microsomes. ........24

3-2 Kinetic parameters (Mean + SD) for the sulfonation of various xenobiotics by
polar bear liver cytosol, listed in order of decreasing enzymatic efficiency. ...........27

4-1 Estimated kinetic parameters (mean & S.D.) for the co-substrate UDPGA in the
glucuronidation of three different OH-PCBs. ...........__......_ ................44

4-2 Kinetic parameters (Mean & S.D.) for the glucuronidation of 4-OHBP and OH-
PCBs. ........... ..... .. ...............48....

4-3 Comparison of the estimated kinetic parameters for OH-PCB glucuronidation in
catfish liver and proximal intestine .............. ...............48....

4-4 Comparison of kinetic parameters (Mean & SEM) for the glucuronidation of
OH- PCBs grouped according to the number of chlorine atoms flanking the
phenolic group ........._.__...... ..__ ...............49....

4-5 Results of regression analysis performed in order to investigate the relationship
between the glucuronidation of OH-PCBs by catfish proximal intestine and liver
and various estimated physical parameters. ............. ...............52.....

5-1 5' 3' Sequences of degenerate primers chosen. ......___ ... ...... ..............66

5-2 Primer pairs chosen, showing annealing temperature and estimated amplicon
length ................. ...............67.................

5-3 Results of BLASTn search of cloned putative partial UGT sequences ........._........78

5-4 Gene-specific primers used in initial 5'RLM-RACE study. ................ ................82

5-5 Gene-specific primers used in succeeding RLM-RACE study ............... .... ............83

5-6 Primers used for amplifying liver and intestinal UGT gene .............. ..................87











5-7 Results of blastn search for livUGTn (and intUGTn) ................ ......................92

5-8 Promoter prediction ................. ...............92........... ....

5-9 Results of blastp search for liv/intUGTp ................. .....__ ............. ......9

5-10 Results of blastn search for I35R C............... ...............99...

5-11 Potential antigenic sites on liv/intUGTp. ............. ...............101....

5-12 Results of blastp search for I3SRCp ................. ...............102........... .

5-13 Results of ClustalW multiple sequence alignment analysis of the cloned UGTs
and the original livUGTn .............. ...............106....

5-14 Conserved consecutive residues observed in catfish liver and mammalian UGTs
(sequences shown in Figure 5-13)............... ...............109.

6-1 UDPGA concentrations (C1M) in liver and intestine of various species ................. 117

6-2 Elution times of certain physiological substances (standards dissolved in mobile
phase) using the anion-exchange HPLC conditions described above...................124

6-3 UDPGA concentrations in CIM (duplicates for individual fish), in catfish liver
and intestine ................. ...............126................

















LIST OF FIGURES


Figure pg

1-1 Schematic of select xenobiotic (represented by hydroxynaphthalene)
biotransformation pathways in the mammalian cell. ............. ....................3

2-1 Structure of the co-substrates PAPS and UDPGA (transferred moieties shown in
bold) and the formation of the polar sulfonate and glucuronide conjugates,
shown here competing for the same substrate ................. .............................6

2-2 Proposed structure of UGT, based on amino acid sequence .............. ..............7

2-3 Complete human UGT1 complex locus represented as an array of 13 linearly
arranged first exons. ............. ...............10.....

2-4 The human UGT2 family. ............. ...............10.....

3-1 Structures of sulfonation sub states investigated in thi s study ................. ...............1 5

3-2 Sulfonation of 3 -OH-B[a]P at PAPS = 0.02 mM ................. ................ ...._..25

3-3 Eadie-Hofstee plot for the glucuronidation of 10 CIM 3-OH-B[a]P, over a
UJDPGA concentration range of 5-3000 CIM. ............. ...............26.....

3-4 Sulfonation of 4'-OH-PCB79, PAPS = 0.02 mM. ................... ............... 2

3-5 Autoradiogram showing the reverse-phase TLC separation of sulfonation
products of OHMXC ................ ...............29........... ....

3-6 Autoradiogram showing the reverse-phase TLC separation of sulfonation
products from incubations with TCPM ................. ...............30........... ...

3-7 Autoradiogram showing the reverse-phase TLC separation of sulfonation
products of TCPM and the effect of sulfatase treatment............._. .. ........._._ ...31

3-8 Autoradiogram showing reverse-phase TLC separation of sulfonation products
from the study of PCP kinetics ....__. ................. ...............32. ...

4-1 Structure of sub states used in channel catfi sh glucuronidation study ................... ..42










4-2 UDPGA glucuronidation kinetics in 4 catfish............... ...............46

4-3 Representative kinetics of the glucuronidation of OH-PCB s in 4 catfi sh. ...............47

4-4 Decrease in Vmax with addition of second chlorine atom flanking the phenolic
group, while keeping the chlorine substitution pattern on the nonphenolic ring
constant ................. ...............50.................

4-5 Relationship between Vmax for OH-PCB glucuronidation in intestine and liver
and ovality .............. ...............53....

5-1 Summary of methods used to clone channel catfish UGT ..........__..................63

5-2 Products of PCR reaction. 1(from intestine), 2 and 3 (from liver) ................... ........75

5-3 Plasmid DNA obtained from cultures transformed with vector containing inserts
from liver and intestine. ............. ...............76.....

5-4 Product of ecoRI digest of purified plasmids containing liver inserts L1-L8..........77

5-5 5'- RLM-RACE and 3'- RACE ................. ........._._......... ................80

5-6 Primer positions for 5'- and 3'-RACE ......... ........ ................ ...............81

5-7 Full nucleotide sequence obtained for hepatic catfish UGT (livUGTn), derived
from 4 sequencing runs each. .............. ...............90....

5-8 Sizes and positions of partial UGT sequences (cross-hatched rectangles) from
intestine and liver, corresponding to two distinct isoforms, relative to complete
sequences for liver and intestinal UGT (solid rectangles). ............. ....................91

5-9 Identification of open reading frame using ORF Finder ................. ............... ....93

5-10 Predicted protein sequence liv/intUGTp from liv/intUGTn .............. ..................93

5-11 Comparison ofliv/intUGTp with homologous proteins in other fish, showing
scores and alignment of closely related sequences. ............. .....................9

5-12 Phylogram for fish UGT proteins homologous to liv/intUGTp .............. ..............96

5-13 Alignment of liv/intUGTp (excluding UTRs) with selected mammalian UGT
proteins, showing scores and multiple alignment of sequences, highlighting
important regions and residues (see discussion) .............. ...............97....

5-14 Phylogram for I.punctatus liv/intUGTp and selected mammalian UGT proteins ...98

5-15 Multiple sequence alignment between livUGTn and I35RC. ............. .................99

5-16 Results of NCBI conserved domain search ................. ...............100........... .










5-17 Kyte-Doolittle Hydrophobicity Plot for liv/intUGTp .............. .....................0

5-18 Results of NCBI conserved domain search for I35R Cp .............. ...................103

5-19 Alignment of predicted protein sequences from cloned catfish UGTs. Regions of
interest and the starting and ending residue of the mature product are
highlighted. ........... ..... ._ ...............104...

5-20 Cloning of livUGTn. ..........._.....__ .....__ .....__ ........... ....105

5-21 Cloning of intUGTn. ..........._.....__ .....__ .....__ ........... ....105

5-22 Multiple sequence alignment for fish sequences homologous to catfish UGT
isolated from liver and intestine, showing regions where substrate binding of
phenols is thought to occur for mammalian UGTI1A isozymes. ................... .........110

5-23 Results of 3' RACE performed on liver, showing multiple products obtained...... 111

5-24 3' RACE for I4. ................ ...............112..............

5-25 PCR amplification of UGT using degenerate primers. ........._._ ... ......_._.......1 14

6-1 Heat-induced degradation of UDPGA (boiling in 0.25 M H2PO4 buffer) ............120

6-2 Decomposition of UDPGA to UDP and UMP after boiling in 0.25 M H2PO4
buffer for 10 minutes ..........._ _..... .._ ...............120.

6-3 Effect of boiling liver tissue for 1 minute in two different concentrations of
buffer. A, 0.25 M H2PO4, pH 3.4; B, 0.30 M H2PO4, pH 4.3 ................ ............... 121

6-4 HPLC chromatogram for catfish AT17 liver. Center refers to region of liver
from which the sample was taken. ............. ...............122....

6-5 HPLC chromatogram for catfish AT18 intestine. Rep 2 refers to second sample
taken from AT18 intestine. .........__ _............ ...............123.

6-6 HPLC chromatogram of UDP, UDP-galacturonic acid (UDPGTA), and UDPGA
standards ................. ...............125................

6-7 Comparison of hepatic and intestinal [UDPGA] in 4 individual channel catfish. .126
















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PHASE II BIOTRANSFORMATION OF XENOBIOTICS BY POLAR BEAR (Grsus
maritimus) AND CHANNEL CATFISH (Ictalurus punctatus)

By

James C. Sacco

August 2006

Chair: Margaret O. James
Major Department: Medicinal Chemistry

Both polar bears and channel catfish are subject to bioaccumulation of persistent

toxic environmental pollutants including hydroxylated compounds, which are potential

substrates for detoxification via phase II conjugative processes such as sulfonation and

glucuronidation. The objectives of this dissertation were to (a) study the capability of

polar bear liver to sulfonate a structurally diverse group of environmental chemicals, and

to study the glucuronidation of 3-OH-B[a]P; (b) study the effects of chlorine substitution

pattern on the glucuronidation of polychlorinated biphenylols (OH-PCBs) by catfish liver

and proxi mal inte sti ne; (c) clone UDP -glucurono syltran sferase (UGT) from catfish liver

and intestine; (d) develop a method to determine physiological concentrations of UDP-

glucuronic acid (UDPGA) in catfish liver and intestine.

In the polar bear, the efficiency of sulfonation decreased in the order 3-OH-

B[a]P>>>triclosan>>4'-OH-PCB79>OHMXC>4'-OH-C165>TCPM>4'-OH-PCB 159

>PCP, all of which produced detectable sulfate conjugates. Substrate inhibition was









observed for the sulfonation of 3-OH-B[a]P and 4'-OH-PCB79. The hexachlorinated

OH-PCBs, TCPM and PCP were poor substrates for sulfonation, suggesting that this may

be one reason why these substances and structurally similar xenobiotics persist in polar

bears .

OH-PCBs are glucuronidated with similar efficiency by channel catfish liver and

proximal intestine. There were differences in the UGT activity profile in both organs.

Both hepatic glucuronidation and intestinal glucuronidation were decreased with the

addition of a second chlorine atom flanking the phenolic group, which is an arrangement

typical of toxic OH-PCBs that persist in organisms.

One full length UGT from catfish liver, together with a full-length UGT (identical

to the liver UGT), and a partial sequence of a different UGT from catfish intestine were

cloned. The full-length catfish UGT clone appeared to be analogous to mammalian

UGTIAl or UGTIA6.

The anion-exchange HPLC method developed to determine UDPGA was sensitive,

reproducible and displayed good resolution for the co-substrate. The hepatic UDPGA

levels determined by this method were similar to those in other mammalian species and

higher than reported for two other fish species. This was the first time intestinal UDPGA

concentrations in any piscine species were determined; the values were similar to rat

intestine, but significantly higher than in human small intestine.















CHAPTER 1
BIOTRANSFORMATION AND ITS IMPORTANCE INT THE DETOXIFICATION OF
XENOBIOTIC S

The exposure of biological systems to environmental compounds which may be

potentially toxic to these systems has spurred the evolution of an elaborate, protective

biochemical system whereby these xenobiotics are eliminated from cells and whole

organisms, usually via chemical transformation (or biotransformation). This system is

composed of a multitude of enzymes, which while being distributed in many tissues and

organs, are principally located in organs such as liver, intestine and lungs. This is of

physiological significance since these tissues represent major routes of xenobiotic entry

into organisms. Within cells, biotransformation enzymes also display a level of

organization in that while some are soluble and found in the cytosol (e.g.

sulfotransferases (SULT), glutathione-S-transferases), others are relatively immobile and

membrane-bound (e.g. UDP-glucuronosyltransferases (UGT) and cytochrome P450s

(CYP) in the endoplasmic reticulum).

Since it is highly improbable that the organism has a substrate-specific enzyme for

metabolizing every potential xenobiotic, biotransformation enzymes are generally non-

specific, acting on a broad range of structurally unrelated substrates. In addition, several

isoforms of the same enzyme (or more than one enzyme) may catalyze product formation

from the same substrate, albeit at different rates and with different affinities. Enzymes in

the same superfamily as those that act upon xenobiotics can also biotransform

endogenous substances, indicating an equally important regulatory role for these










enzymes. This interrelationship between different enzymes and substrates can be

illustrated by the metabolism of P-estradiol in humans, which can be biotransformed both

via sulfonation (SULTIE1, which also acts on 7-hydroxymethyl-12-dimethylbenz-

anthracene, the product of CYP450-catalyzed hydroxylation of 7, 12-dimethyldibenz-

anthracene (Glatt et al., 1995)) and glucuronidation (UGTIA1, which can also conjugate

1-naphthol (Radominska-Pandya et al., 1999)).

While these enzymes mainly represent a cellular defense mechanism against

toxicity, occasionally procarcinogenic and protoxic xenobiotics are metabolized to active

metabolites that attack macromolecules such as DNA, proteins and lipids.

In exposed organisms, metabolism is an important factor in determining the

bioaccumulation, fate, toxicokinetics, and toxicity of contaminants. The majority of the

compounds of interest to this study are derived from Phase I metabolism of

environmental pollutants. These metabolites have been shown to have toxic effects both

in vitro and in vivo, effects that can be eliminated by Phase II biotransformation (Chapter

2). In addition, contaminant exposure can result in the induction or inhibition of both

Phase I and Phase II enzymes. For example, induction of CYP 1A (e.g., by polyaromatic

hydrocarbons (PAHs) or co-planar polychlorinated biphenyls (PCBs)), CYP 2B and

CYP3A (e.g., by o-chlorine substituted PCBs) will lead to increased formation of

hydroxylated metabolites. Thus, a balance between the CYP and conjugative Phase II

enzymes, sometimes directly mediated by the xenobiotic substrates and/or their

metabolites, is responsible for either the detoxification or the accumulation of toxic

metabolites in the body. The final removal of these metabolites from the cell is brought

about by several different groups of membrane proteins (e.g., organic anion transport










protein (OATP), multidrug-resistance associated protein (MRP)), a process sometimes


referred to as Phase III biotransformation (Figure 1-1).


MRP
OA TP


' OH


MRP
OA TP


'"r
oHOH


I I


\ cytosol ER membrane ER lumen
Figure 1-1. Schematic of select xenobiotic (represented by hydroxynaphthalene)
biotransformation pathways in the mammalian cell. For abbreviations see text.


I















CHAPTER 2
PHASE II CONJUGATION: GLUCURONIDATION AND SULFONATION

Biotransformation has been conveniently categorized into two distinct phases.

While the consecutive numbering of these processes implies a sequence, this is not

always the case and the extent of involvement of both phases in the metabolism of a

compound depends on both its chemical structure and physical properties. Phase I

biotransformation usually consists of oxidations carried out largely by CYP enzymes and

flavin monooxygenases and hydrolysis reactions executed by ester hydrolase, amidase

and epoxide hydrolase (EH). A variety of chemical moieties can be conjugated to suitable

acceptor groups on xenobiotics as part of Phase II biotransformation, including

glucuronic acid (UGT), sulfonic acid (SULT), glutathione (GST), amino acids, and an

acetyl group (N-acetyltransferase) .

With the exception of acetylation, methylation and fatty acid conjugation, the

strategy of Phase II biotransformation is to convert a xenobiotic to a more hydrophilic

form via the attachment of a chemical moiety which is ionizable at physiological pH. The

resulting anionic conjugate is then readily excreted in bile, feces, or urine, and is

generally unable to undergo passive penetration of cell membranes. This metabolic

transformation also results in reduced affinity of the compound for its cellular target.

Enterohepatic recycling may result in the hydrolysis of biliary excreted conjugates and

the regeneration of the parent compound, which is then subj ect again to

biotransformation after being reabsorbed through the gut mucosa. In a few cases, the










conjugate is pharmacologically active, as in the case of morphine-6-glucuronide

(Yoshimura et al., 1973) and minoxidil sulfate (Buhl et al., 1990).

The moieties attached to the xenobiotic in the case of sulfonation and

glucuronidation are a sulfonate group (pKa 2) or glucuronic acid (pKa 4-5). The co-

substrates which supply these highly polar species are, respectively, 3'-phosphoadenosyl-

5'-phosphosulfate (PAPS) and uridine 5' -diphosphoglucuronic acid (UDPGA) (Figure 2-

1). The mechanism of both reactions, which occurs as a ternary complex, is a SN2

reaction, the deprotonated acceptor group of the substrate attacking the sulfur in the

phosphosulfate bond of PAPS, or the C1 of the pyranose ring to which UDP is attached in

an ot-glycosidic bond in the case of UDPGA. The resulting conjugates are then released.

PAP and UDP also leave the enzyme's active site and are subsequently regenerated.

There may be competition for the same acceptor group, especially for phenols.

Other acceptor groups that can be conjugated by both processes include alcohols,

aromatic amines and thiols. Glucuronidation is also active on other functional groups,

including carboxylic acids, hydroxylamines, aliphatic amines, sulfonamides and the C2 Of

1 ,3 -di carb onyl compounds. SULTs are generally high-affinity, low-capacity

biotransformation enzymes that operate effectively at low substrate concentrations. Thus,

typical Knas for the sulfonation of xenobiotic substrates are usually significantly lower

than Knas for the same substrates undergoing biotransformation by the low-affinity, high-

capacity UGTs. For example, kinetic parameters for the sulfonation and glucuronidation

of the antimicrobial agent triclosan in human liver are Km values of 8.5 and 107 C1M and

Vmax of 96 and 739 pmol/min/mg protein respectively (Wang et al., 2004).

















OH OH/
o P P O

'OHH

UDPGA


PAPS


PAP


SULT (cytosol)





~UG T (ER)


sulfonate conjugate






\ 0,0




Cl OH


xenobiotic


UDP


glucuronide conjugate



Figure 2-1. Structure of the co-substrates PAPS and UDPGA (transferred moieties shown
in bold) and the formation of the polar sulfonate and glucuronide conjugates,
shown here competing for the same substrate.










UDP-Glucuronosyltransferases (UGTs)

The primary sequence of human UGTs ranges from 529 to 534 amino acids in

length (Tukey and Strassburg 2000). These 50-56 kDa proteins reside in the endoplasmic

reticulum, whereby the amino terminus and around 95% of the subsequent residues are

located in the lumen. A 17-amino acid-long transmembrane segment connects the

lumenal part of the enzyme with the short (19-24 residues) carboxyl-terminus located in

the cytosol (Figure 2-2). The active enzyme probably consists of dimers, linked together

at the C-terminus (Meech and Mackenzie 1997). The existence of tetramers for the

formation of the diglucuronide of B[a]P-3,6-diphenol has been suggested (Gschaidmeier

and Bock 1994).

++
COO. Cytosol

ER
III membrane

ER lumen

NH3+
Ag lycone
UDPGA






Figure 2-2. Proposed structure of UGT, based on amino acid sequence

Based on evolutionary divergence, mammalian UGTs have been classified into four

distinct families (Mackenzie et al., 2005): family 1, which includes bilirubin, thyroxine

and phenol UGTs; family 2, which includes steroid UGTs; family 3, which includes

UGTs whose substrate specificity is, as yet, unknown (Mackenzie et al., 1997); family 8,

represented by UGT8Al which utilizes UDP-galactose as the sugar donor (Ichikawa et










al., 1996). Although the liver is the major site of glucuronidation in the living organism,

several other tissues have been shown to express UGTs. The small intestine appears to be

an equally important site of glucuronidation, particularly for ingested xenobiotics. In

addition, expression of some UGT isoforms is tissue-specific (Table 2-1).

The nine family 1 UGT isoforms (UGT1) are all encoded by one gene that has

multiple unique exons located upstream of four common exons on human chromosome

2q37 (Figure 2-3). The isoforms are generated by differential splicing of one unique first

exon (which encodes two-thirds of the lumenal domain, starting from the N-terminus,

288 amino acids long) to the four common exons (exons 2-5, which encode the remainder

of the lumenal domain, the transmembrane domain and the cytosolic tail, 246 amino

acids long). Due to this unusual gene structure and splicing mechanism, the UGT1

isoforms have variable amino-terminal halves and identical carboxyl-terminal halves.

While the first exon determines substrate specificity, the common exons specify the

interaction with UDPGA (Ritter et al., 1992; Gong et al., 2001). Thus, the major bilirubin

UGT (UGTIAl) of humans, rats and other species is encoded by exon 1 and the adjacent

4 common exons. The phenol UGT (UGTIA6) is encoded by exon 6 and the 4 common

exons.

The human UGT2 gene family includes three members of the UGT2A subfamily

and twelve members of the UGT2B subfamily (Mackenzie et al., 2005). The UGT2

proteins are encoded by separate genes consisting of six exons located on human

chromosome 4ql3. The region of the protein encoded by exons 1 and 2 is equivalent to

that encoded by the unique exons 1 of the UGT1 isoforms, and the subsequent

intron/exon boundaries are in corresponding positions in both gene families. Similar to









the UGTIA enzymes, the UGT2Al and 2A2 proteins have identical C-termini and

different N-termini that arise due to differential splicing of the first exon (Figure 2-4). By

contrast, the UGT2A3 gene comprises six exons that are not shared with the other two.

Table 2-1. Expression of human UGT mRNA in various tissues
UGT Liver Intestine Esophagus Kidney Brain Prostate Other tissues
& stomach
1Al ///
1A3 J J Jb
1A4
1A6 J J Jb J testis, ovary
1A7 c
1A8
1A9
1A10c
2Al Olfactory
epithelium, lung
2B4
2B7 Pancreas
2B10 mammary
gland,
2B11 mammary
gland, adrenal,
skin, adipose
2B15 mammary
gland, adipose,
skin, lung,
testis, uterus,
placenta
2Bl7

a Tukey and Strassburg 2000; King et al., 2000; Lin and Wong 2002; Wells et al., 2004
b only a third of the population expresses these isoforms in gastric epithelium (Strassburg
et al., 1998)
c expressed in bile ducts











5' 3'
Exons 1 C~*ommon
1A12plA11p 1A8 1A10 1A13p 1A9 1A7 1A6 1A51A41A3 1A~plA1
2345



300 kb 218 kb 95 kb

Primary transcripts

Et UGT1A1
L--~~X/CUGT1A8

Isozymes
IIIIIUGT1A1
Etc .
I UGT1A8



Figure 2-3. Complete human UGT1 complex locus represented as an array of 13 linearly
arranged first exons.

Each first exon, except for the defective UGTIAl2p and UGTIAl3p pseudo
ones, contains a 5'proximal TATA box element (bent arrow) that allows for
the independent initiation of RNA polymerase activity that generates a series
of overlapping RNA transcripts (Adapted from Gong et al., 2001).

5' 3'
2B29p 2B17p 2815 2810 2A3 2B27p 2B26p 2B7 2811 2B28 2B25P 2B24P 2B4 2A1/2
I UI I UI I UI U U U U UI U U 1





2A1 2A2 2 3 4 5 6

1 1 11 I l


Figure 2-4. The human UGT2 family.

Each gene (not drawn to scale), consisting of six exons, is represented by a
white rectangle, except for '2A1/2', which represents seven exons (1 unique
first exon and shared exons 2-6). Adapted from Mackenzie et al. (2005).









Sulfotransferases (SULTs)

Sulfotransferases can be either membrane-bound in the Golgi or in the cytosol.

While the membrane-bound SULTs sulfonate large molecules such as

glucosaminylglycans, the cytosolic enzymes are involved in the inactivation of

endogenous signal molecules (steroids, thyroid hormones, neurotransmitters) and the

biotransformation ofxenobiotics.

Each cytosolic SULT is a single a/p globular protein with a characteristic five-

stranded parallel sheet, with a-helices flanking each sheet. The active enzyme is a

homodimer, with each polypeptide chain having a MW of about 35,000. Kakuta et al.

(1997) were the first group to solve the first X-ray structure for the SULT family. Mouse

estrogen sulfotransferase (mEST) was shown completed with PAP and the substrate

estradiol (E2). The binding of estradiol to human SULTIAl has also been demonstrated

(Gamage et al., 2005). Both PAPS- and substrate-binding sites are located deep in the

hydrophobic substrate pocket. The structures of four human cytosolic enzymes have also

been elucidated : SULT 1Al (Gamage et al., 2003), dop ami ne/c atechol ami ne

sulfotransferase (SULTIA3) (Bidwell et al., 1999; Dajani et al., 1999), hydroxysteroid

sulfotransferase (SULT2Al; hHST) (Pedersen et al., 2000), and estrogen sulfotransferase

(SULTIE1; hEST) (Pedersen et al., 2002).

Five SULT gene families have been identified in mammals (SULTsl-5). While

SULT enzymes have different substrate specificities, the repertoire of suitable substrates

is so broad that it is not uncommon that one substrate is biotransformed by more than one

enzyme. SULTs are distributed in a wide variety of tissues (Table 2-2). In humans, liver

cytosol has been shown to contain mostly SULTslA1, 2A1, and 1E1, with lesser amounts














































4Al J

a reviewed by Glatt 2002.
b mRNA of fetal tissues
Soral mucosa


Using 3 -hydroxy-benzo(a)pyrene (3-OH-B[a]P) and 9-OH-B[a]P, the existence of

multiple SULT isoforms in channel catfish liver and intestine, including a 3-

methylcholanthrene-inducible form of phenol-SULT in liver, has been established

(Gaworecki et al., 2004; James et al., 2001). The phenol-SULT in catfish liver and


of SULTs 1A2, 1B1, 1El and 2Al. While SULTIAl and SULTIE1 are responsible for

most of the phenol and estrogen SULT hepatic activity respectively, SULT2Al

(hydroxysteroid SULT) shows greater affinity for alcohols and benzylic alcohols (Mulder

and Jakoby, 1990; Glatt, 2002).


Table 2-2. Tissue distribution of SULTs (cDNA and mRNA) in humans
SULT Liver Intestine Esophagus Kidney Brain Lung Other tissues
& stomach


J /
J /
/
J /


J /
J J


1Al
1A2
1A3
1B1


IC2


1C4


1El


2Al


2B1


Platelets


J Platelets
J J Spleen, kidney,
leukocytes
J Jb Ovary, spinal
cord, heart
J J Thyroid gland,
ovary
Jb J Jb Endometrium,
skin, mammary
Adrenal gland,
ovary
J J Placenta,
prostate,
platelets









intestine has been isolated as a 41,000 Da protein. A second protein with a molecular

weight of 31,000 Da, isolated from liver, has not been identified to date. Interestingly

enough, SULT activity with phenolic substrates is higher in intestine than liver (Tong and

James 2000). Other hepatic SULTs isolated and characterized from fish include

petromyzonol SULT from lamprey (Petromyzon marinus) larva (which displays 40%

homology with mammalian SULT2Bla, or cholesterol SULT) and a bile steroid SULT

from the shark Heterodontus portusjacksoni (Venkatachalam et al., 2004; Macrides et al.,

1994).















CHAPTER 3
SULFONATION OF XENOBIOTICS BY POLAR BEAR LIVER

The lipophilicity and inherent chemical stability of persistent organic pollutants

(POPs) renders them excellent candidates for absorption through biological membranes

as well as accumulation in both organisms and their environment. Many POPs have been

shown to biomagnify in food webs to potentially toxic levels in top predators such as the

polar bear (Grsus maritimus), whose diet mainly consists of ringed seal (Phoca hispida)

blubber (Kucklick et al., 2002).

Since the sulfonation of xenobiotics has never been studied in the polar bear, the

obj ective of this study was to investigate the efficiency of this route of detoxification on a

select group of known environmental pollutants: 4'-hydroxy-3,3',4,5 '-

tetrachlorobiphenyl (4'OH-PCB79), 4'-hydroxy-2,3,3 ',4,5,5 '-hexachlorobiphenyl (4'-

OH-PCBl159), 4'-hydroxy-2,3,3 ',5,5 ',6-hexachlorobiphenyl (4'-OH-PCBl165),

pentachlorophenol (PCP), tris(4-chlorophenyl)-methanol (TCPM), 2-(4-methoxyphenyl)-

2-(4-hydroxyphenyl)- 1,1,1 -trichloroethane (OHMXC), 3 -hydroxybenzo(a)pyrene (3-OH-

B [a]P), triclosan (2,4,4'-trichloro-2 '-hydroxydiphenyl ether) (Figure 3-1). The OH-PCBs

were named as PCB metabolites, according to the convention suggested by Maervoet et

al. (2004).

Polychlorinated biphenylols (OH-PCBs), major biotransformation products of

PCBs (James, 2001), have been shown to be present in relatively high concentrations in

polar bears (Sandau and Norstrom 1998; Sandau et al., 2000). The abundance of these

hydroxylated metabolites may be due to CYP induction (Letcher et al., 1996), inefficient








































(5) (6)


Figure 3-1. Structures of sulfonation substrates investigated in this study.

(1) 3-OH-B[a]P; (2) triclosan; (3) 4'-OH-PCB79; (4) 4'-OH-PCBl159; (5) 4'-OH-
PCBl65; (6) OHMXC; (7) TCPM; (8) PCP. Full names of each compound are
given in the text.


CI









CI OH









Phase II detoxication, and inhibition of their own biotransformation. The 4'-OH-PCB79

(an oxidation product of PCB congener 77) is a potent inhibitor of the sulfonation of

several substrates, including 3-OH-B[a]P in channel catfish intestine and human liver

(van den Hurk et al., 2002, Wang et al., 2005), 4-nitrophenol by human SULTIAl (Wang

et al., 2006), 3,5-diiodothyronine (T2) in rat liver (Schuur et al., 1998), and estradiol by

human SULTIE1 (Kester et al., 2000). Both 4'-OH-PCBl59 and 4'-OH-PCBl65 have

been shown to inhibit the sulfonation of 3-OH-B[a]P and 4-nitrophenol by human SULT

(Wang et al., 2005, 2006). Another compound detected in polar bears is PCP (Sandau and

Norstrom 1998), a commonly used wood preservative that has been implicated in thyroid

hormone disruption in Arctic Inuit populations (Sandau et al., 2002). TCPM is a globally

distributed organochlorine compound of uncertain origin, which was reported in human

adipose tissue (Minh et al., 2000). Polar bear liver contains 4000-6800 ng/g lipid weight

TCPM, the highest levels recorded for this compound in all species studied (Jarman et al.,

1992). TCPM is a potent androgen receptor antagonist in vitro (Schrader and Cooke

2002). OHMXC, formed by demethylation of the organochlorine pesticide methoxychlor,

is an estrogen receptor (ER) oc agonist, an ERP antagonist and an androgen receptor

antagonist (Gaido et al., 2000). The ubiquitous environmental pollutant benzo[a]pyrene is

mainly metabolized to 3-OH-B[a]P, a procarcinogen that can be eliminated via

sulfonation (Tong and James 2000). Together with its 7,8-dihydrodiol-9, 10-oxide and

7,8-oxide metabolites, 3-OH-B[a]P can form adducts with macromolecules and initiate

carcinogenesis (Ribeiro et al., 1986). Triclosan is an antimicrobial agent that has been

detected in human plasma and breast milk (Adolfsson-Erici et al., 2002). In vitro studies









have shown that triclosan inhibits various biotransformation enzymes, including SULT

and UDP-glucuronosyltransferases (UGT) (Wang et al., 2004).

The fact that 3-OH-B[a]P, triclosan, OHMXC, 4'-OH-PCB79, 4'-OH-PCBl59 and

4'-OH-PCBl165 have not been reported as environmental contaminants in polar bears to

date may be due to non-significant levels in the Arctic environment or efficient

metabolism via, for example, sulfonation. On the other hand, the presence of PCP and,

particularly, high amounts of TCPM in these Arctic carnivores, may indicate poor

sulfonation of these substrates. The polychlorobiphenylols 4'-OH-PCBl59 and 4'-OH-

PCBl165 are of interest since though they have not been detected in polar bears, they are

structurally similar to 4'-OH-PCBl72, one of the major OH-PCBs found in polar bear

plasma (Sandau et al., 2000). It is thus possible that these compounds are sulfonated with

similar efficiencies. The other major Phase II biotransformation pathway for the above-

mentioned compounds is glucuronidation. Polar bear liver efficiently glucuronidated 3-

OH-B[a]P and several OH-PCBs (Sacco and James 2004).

Hypothesis

Sulfonation occurring in polar bear liver is an inefficient route of detoxification for

a structurally diverse group of environmental contaminants.

Methodology

Unlabeled PAPS was purchased from the Dayton Research Institute (Dayton, OH).

Uridine 5' -diphosphoglucuronic acid (UDPGA) was obtained from Sigma (St.Louis,

MO). Radiolabeled [35S]PAPS (1.82 or 3.56 Ci/mmol) was obtained from Perkin-Elmer

Life Sciences, Inc. (Boston, MA). The benzo[a]pyrene metabolites 3-OH-B[a]P, B[a]P-3-

O-sulfate and B [a]P-3-O-glucuronide were supplied by the Midwest Research Institute

(Kansas City, MO), through contact with the Chemical Carcinogen Reference Standard










Repository of the National Cancer Institute. Dr. L.W.Robertson, U of Iowa, kindly

donated the 4'-OH-PCB79, and 4'-OH-PCBl59 and 4'-OH-PCBl65 were purchased

from AccuStandard, Inc. (New Haven, CT). PCP from Fluka Chemical (Milwaukee, WI)

was used to prepare the water-soluble sodium salt (Meerman et al., 1983). Triclosan and

sulfatase (Type VI from Aerobacter, S1629) were purchased from Sigma (St.Louis, MO),

while methoxychlor and TCPM were purchased from ICN Biomedical (Aurora, OH) and

Lancaster Synthesis, Inc. (Pelham, NH), respectively. The OHMXC was prepared by the

demethylation of methoxychlor and purified by recrystallization (Hu and Kupfer 2002).

Tetrabutyl ammonium hydrogen sulfate (PIC-A low UV reagent) was from Waters

Corporation, Milford, MA. Other reagents were the highest grade available from Fisher

Scientific (Atlanta, GA) and Sigma.

Animals. The samples used in this study were a kind donation from Dr. S. Bandiera (U

British Columbia) and Dr. R. Letcher (Environment Canada). They were derived from

the distal portion of the right lobe of livers of three adult male bears G, K and X. Bears G

and K were collected as part of a legally-controlled hunt by Inuit in the Canadian Arctic

in April 1993 near Resolute Bay, Northwest Territories, while bear X was collected in

November 1993 near Churchill, Manitoba, just after the fasting period. Liver samples

were removed within 10-15 minutes after death, cut into small pieces and frozen at -

196oC in liquid N2. The samples were subsequently stored at -80oC.

Cytosol and Microsomes Preparation. Prior to homogenization, the frozen polar

bear liver samples (~2g) were gradually thawed in a few ml of homogenizing buffer.

Homogenizing buffer consisted of 1.15% KC1, 0.05 M K3PO4 pH 7.4, and 0.2 mM

phenylmethylsulfonyl fluoride, added from concentrated ethanol solution just before use.









Resuspension buffer consisted of 0.25 M sucrose, 0.01 M Hepes pH 7.4, 5% glycerol, 0. 1

mM dithiothreitol, 0.1 mM ethylene diamine tetra-acetic acid and 0.1 mM phenylmethyl

sulfonyl fluoride. The liver was placed in a volume of fresh ice-cold buffer equal to 4

times the weight of the liver sample. The cytosol and microsomal fractions were obtained

using a procedure described previously (Wang et al., 2004). Microsomal and cytosolic

protein contents were measured by the Lowry assay, using bovine serum albumin (BSA)

as standard.

Sulfotransferase Assays

A. Fluorometric method. The activity was measured on the basis that at alkaline pH, the

benzo[a]pyrene-3 -O-sulfate has different wavelength optima for fluorescence excitation

and emission (294/415 nm) from the benzo[a]pyrene-3 -O-phenolate anion (3 90/545 nm)

(James et al., 1997). Saturating concentrations of PAPS were determined by performing

the assay at 1 C1M 3-OH-B[a]P. The reaction mixture for detecting the sulfation of 3-OH-

BaP by polar bear liver cytosol consisted of 0.1 M Tris-Cl buffer (pH 7.6), 0.4% BSA,

PAPS (0.02 mM), 25 Clg polar bear hepatic cytosolic protein, and 3-OH-B[a]P (0.05-25

CIM) in a total reaction volume of 1.0 mL. SULT activity (pmol/min/mg) was calculated

from a standard curve prepared with B[a]P-3-O-sulfate standards. Substrate consumption

did not exceed 10%.

B. Radiochemical extraction method. This method, based on Wang and co-workers

(2004), was employed in the study of the sulfonation of 4'-OH-PCB79, 4'-OH-PCBl159,

4'-OH-PCBl65, triclosan, PCP, TCPM and OHMXC. Cytosolic protein concentrations

and incubation time were optimized for every test substrate to ensure that the reaction

was linear during the incubation period. Substrate consumption did not exceed 5%. The









incubation mixture consisted of 0.1 M Tris-Cl buffer (pH 7.0), 0.4% BSA in water, 20

CIM PAPS (10% labelled with 35S), 0.1 mg polar bear hepatic cytosolic protein, or 0.005

mg in the case of 4'-OH-PCB79 and triclosan, and substrate in a total reaction volume of

0.1 mL, or 0.5 mL in the case of TCPM. The OH-PCBs, triclosan and OHMXC were

added to tubes from methanol solutions, and the methanol was removed under N2 priOr to

addition of other components. The TCPM was dissolved in DMSO, the solvent being

present at a concentration not exceeding 1% in the final assay volume. Control

determinations utilizing 1% DMSO had no inhibitory effect on sulfonation. Aqueous

solutions of sodium pentachlorophenolate were utilized in the case of PCP. Tubes

containing all components except the co-substrate were placed in a water bath at 37oC

and PAPS was added to initiate the reaction. Incubation times were 5 min (TCPM), 20

min (4'-OH-PCB79, triclosan), 30 min (PCP) and 40 min (OHMXC, 4'-OH-PCBl59, 4'-

OH-PCBl165). The incubation was terminated by the addition of an equal volume of a 1:1

mixture of 2.5% acetic acid and PIC-A and water. The sulfated product was extracted

with 3.0 mL ethyl acetate as described previously (Wang et al., 2004) and the phases

were separated by centrifugation. Duplicate portions of the ethyl acetate phase were

counted for quantitation of sulfate conjugates.

C. Radiochemical TLC method. Since the ethyl acetate phase contains sulfate

conjugates formed from both the substrate of interest and substrates already present in

polar bear liver, TLC was used to quantify substrate sulfation in cases where SULT

activity was similar in samples and substrate blanks. After evaporating 2 ml of ethyl

acetate extract from the SULT assay under N2, the solutes were reconstituted in 40 C1L

methanol. For 4'-OH-PCBl59, 4'-OH-PCBl65, PCP and OHMXC, the substrate










conjugates were separated on RP-18F254s TOVeTSe phase TLC plates with fluorescent

indicator (Merck, Darmstadt, Germany) using methanol:water (80:20). For TCPM,

Whatman KClsF reverse phase 200 Clm TLC plates with fluorescent indicator in

conjunction with a developing solvent system consisting of methanol:water:0.28 M PIC-

A (40:60:1.9 by volume) were employed. Electronic autoradiography (Packard Instant

Imager, Meriden, CT) was used to identify and quantify the radioactive bands separated

on the TLC plate. The counts representing the substrate sulfate conjugate products were

expressed as a fraction of the total radioactivity determined by scintillation counting, thus

enabling the radioactivity due to the substrate conjugate to be accurately determined.

The identity of the conjugate of TCPM as a sulfate ester was verified by studying

its sensitivity to sulfatase. Polar bear cytosol (0.5 mg) was incubated for 75 minutes with

or without 200 CLM TCPM. The incubation was terminated, and the product extracted into

ethyl acetate as above. The ethyl acetate was evaporated to dryness and dissolved in 0.25

mL of Tris buffer, pH 7.5, containing 0 or 0.08 units of sulfatase. Following an overnight

incubation at 35oC, the reaction was stopped by the addition of methanol and the tubes

were centrifuged. The supernatants were evaporated to dryness, reconstituted in methanol

and analyzed by TLC as described above.

UDP-Glucuronosyltransferase Assay. The reaction mixture for detecting the

glucuronidation of 3-OH-B[a]P by polar bear liver microsomes consisted of 0. 1 M Tris-

HCI buffer (pH 7.6), 5 mM MgCl2, 0.5% Brij-58, UDPGA (4 mM), 5 Clg polar bear

hepatic microsomal protein, and 3-OH-B[a]P in a total reaction volume of 500 CIL. The

substrate, 3-OH-B[a]P in methanol, was blown dry under N2 in the dark in a tube to

which, after complete evaporation, a premixed solution of microsomal protein and Brij-









58 (in a 5:1 ratio) was added, vortexed, and left for 30 minutes on ice. Subsequently, the

buffer and water were added in that order and vortex-mixed. Immediately preceding a 20-

minute incubation at 37oC, UDPGA was added to initiate the reaction. The reaction was

terminated by the addition of 2 mL ice-cold methanol. Precipitated protein was pelleted

by centrifugation at 2000 rpm for 10 minutes. The supernatant, 2 mL, was then mixed

with 0.5 mL NaOH (lN) and the fluorescence of B[a]P-3-glucuronic acid measured at

excitation/emission wavelengths of 300/421 nm (Singh & Wiebel, 1979). The activity of

UGT (nmol/min/mg) was then determined.

Preliminary studies established the conditions for linearity of reaction with respect

to time, protein and detergent concentrations, at the same time ensuring that substrate

consumption did not exceed 10%. The apparent Km for UDPGA was determined by

performing experiments at a fixed concentration of 3-OH-B[a]P (10 C1M). Saturating

UDPGA concentrations were used in order to determine 3-OH-B[a]P glucuronidation

kinetics.

Kinetic Analysis. Duplicate values for the rate of conjugate formation at each substrate

concentration were used to calculate kinetic parameters using Prism v 4.0 (GraphPad

Software, Inc., San Diego, CA). Equations used to fit the data were the Michaelis-Menten

hyperbola for one-site binding (eq. 1), the Hill plot (eq. 2), substrate inhibition for one-

site binding (eq. 3) (Houston and Kenworthy 2000), and partial substrate inhibition due to

binding at an allosteric site (eq. 4) (Zhang et al., 1998).

v = Vmax[S] / (Km + [S]) (1)

v = Vmax[S]h / (S50h + [S]h) (2)

v = Vmax[S] / (Km + [S] + ([S]2/K,)) (3)









v = Vmaxy(1 + (Vmax2[S]/Vmax;K,)) / (1 + Km/[S] + [S]/K,) (4)

Values for K, and Vmax derived from equation 1 were used as initial values in the

fitting of data to equations 3 and 4. Eadie-Hofstee plots were used in order to analyze the

biphasic kinetics observed.

Results

Sulfonation and glucuronidation of 3-OH-B[a]P

Optimum conditions for sulfonation were 10 minutes incubation time and 25 Cpg

cytosolic protein. A concentration of 0.02 mM PAPS provided saturating concentrations

of the co-substrate and enabled kinetic parameters at 1.0 CLM 3-OH-B [a]P to be calculated

by the application of eq. 1 (Table 3-la). The data for the sulfonation of 3-OH-B[a]P was

fit to a two-substrate model (eq. 3), whereby the binding of a second substrate to the

enzyme is responsible for the steep decline in enzyme activity at concentrations

exceeding 1 CLM (Figure 3-2a). Initial estimates of Vmaxi and Km were provided by the

initial data obtained at low [S] (non-inhibitory), while Vmax2 WAS constrained to 65 + 20

pmol/min/mg, which is slightly below the plateau in Figure 3-2a.

The kinetic scheme (Figure 3-2b) illustrates the proposed partial substrate

inhibition process, which assumes that substrate binding is at equilibrium, which is

probable due to the low turnover rate of SULT. The best fit of the data was provided by a

K, of 1.0 + 0.1 CLM. Binding of the second substrate molecule results in a tenfold

reduction in the rate of sulfonate formation.





































a constrained variables to obtain best fit
b ValUeS for high-affinity component
c values for low-affinity component


Table 3-1. Estimated kinetic parameters (Mean f SD) for (a) sulfonation and (b) glucuronidation of 3-OH-B[a]P by polar bear liver
cytosol and microsomes. Values were calculated as described in the Methodology.


(a) sulfonation

Substrate Vmax1 (app)

(pmol/min/mg)

3-OH-B[a]P 500 f 8

PAPS 162 f 35

(b) glucuronidation

Sub state Vmax (app)


Km (app)

(cLM)

0.41 f 0.03

0.22 f 0.07


Vmaxl/Km Vmax2 (app) a

(CIL/min/mg) (pmol/min/mg)

1220 f 70 65.0 f 20.0


Kz (app)

(cLM)

1.01 & 0.10


Vmax2/K,

(CLL/min/mg)

66.2 f 26.8


(nmol/min/mg)

3.00 f 1.18

1.53 f 0.56b, 1.47 f 0.48c


Km (app)

(cLM)

1.4 f 0.2

42.9 f 2.5b, 200 f 68c


Vmax/ Km

(CLL/min/mg)

1900 f 544


3 -OH-B [a]P

UDPGA
















(a)

300- PB G
I PB K
liii v PB X
E 200-







0 10 20 30

[3-OH-B(a)P] ( CMh)

(b)

PAPS K,, 10.4 IIM) PAPS V,,avy (471.8 pmol/min/mg)
E E
30HBaP

1K, (1.2 p M)


E PPSV,,,, (45 .0 pmnol'mninl'm g)
(30HBaP)2


Figure 3-2. Sulfonation of 3-OH-B[a]P at PAPS = 0.02 mM.

A. Each data point represents the average of duplicate assays for each bear,
while the error bars represent the standard deviation. The line represents the
best fit to the data of equation (3). B) Kinetic model for partial substrate
inhibition of SULT by 3-OH-B[a]P, after Zhang et al. (1998). E refers to
SULT .









Optimum conditions for the glucuronidation of 3-OH-B[a]P by polar bear

microsomes were found to be 5 Cpg microsomal protein and a 20-minute incubation. A

concentration of 4 mM UDPGA was determined to be suitable for providing saturating

concentrations of the co-substrate. The binding of UDPGA to UGT at 10 CLM 3-OH-

B[a]P was shown to be biphasic, with a fivefold reduction in affinity at higher UDPGA

concentrations (Table 3-1b). The kinetic parameters for the co-substrate were calculated

by deconvoluting the curvilinear data in the Eadie-Hofstee plot (Figure 3-3). In the

presence of 4 mM UDPGA, the formation of B[a]P-3-O-glucuronide followed Michaelis-

Menten kinetics (Table 3-1b).





ow-affinity
3- A high-affinity









0 10 20J 30 40
vi[S]


Figure 3-3. Eadie-Hofstee plot for the glucuronidation of 10 CLM 3-OH-B[a]P, over a
UJDPGA concentration range of 5-3000 CLM.

Each data point represents the average of duplicate assays for all bears, while
the error bars represent the standard deviation.





Sulfonation of other substrates

Triclosan sulfate was formed rapidly, with the overall kinetics conforming to a

hyperbolic curve (eq. 1) (Table 3-2). Substrate inhibition was observed for 4'-OH-PCB79

(Figure 3-4), with the data fitting equation (3). The value of K, that gave the best fit was

217 f 25 CLM (Table 3-2). Sulfate conjugation of 4'-OH-PCBl59 and 4'-OH-PCBl65,

which proceeded via Michaelis-Menten kinetics, was, respectively, 11 and 5 times less

efficient than the sulfonation of 4'-OH-PCB79 (Table 3-2). At a concentration of 10 CLM,

4'-OH-PCBl165 was observed to inhibit sulfonation of substrates already present in polar

bear liver cytosol by 60%.


Table 3-2. Kinetic parameters (Mean f SD) for the sulfonation of various xenobiotics by
polar bear liver cytosol, listed in order of decreasing enzymatic efficiency.
All data fit equation (1), except for 4'-OH-PCB79 and PCP, which fit
equations (3) and (2) respectively (see Methodology for equations).

Substrate Vmax Km Vmax / Km Ki
(pmol/min/mg) (yM) (pL/min/mg) (yM)


triclosan 1008 f 135 11 f 2
4'-OH-PCB79 372 f 38 123 f 20
OHMXC 51.1 f 7.8 67 f 4
4'-OH-PCBl165 8.6 f 2.0 17 f 7
TCPM 62.0 f 11.2 144 f 36
4'-OH-PCBl159 14.8 f 2.3 60 f 21
PCP 13.8 f 1.2 72 f 14b
aK, for bears G, K and X were 240, 220 and 190 LM
constrained to obtain the best fit for the data
bSso; h = 2.0 f 0.4


90.8 f 6.8
3.1 f 0.3 217 f 25a
0.8 f 0.1
0.56 f 0.17
0.44 f 0.06
0.28 f 0.12
0.20 f 0.05
respectively. These values were











I PBK
SPB X











0 100 200 300 4100 500 600 700 800


[4'-OH-PCB79] (CLM)


Figure 3-4. Sulfonation of 4'-OH-PCB79, PAPS = 0.02 mM.

Each data point represents the average of duplicate assays for each bear, while
the error bars represent the standard deviation. The line represents the best fit
to equation (4) for 4'-OH-PCB79.


Due to variable rates of sulfonation of these unknown substrates, autoradiographic

counts corresponding to the OHMXC-O-sulfate band were used to correct the activities

calculated from the scintillation counter data (Figure 3-5). This enabled the transformed

data to be fit into a Michaelis-Menten model (Table 3-2). The autoradiograms obtained

showed that increasing concentrations of OHMXC resulted in decreased counts for the

unknown sulfate conjugates (Figure 3-5). Sulfonation of the unknown substrates in polar

bear cytosol was reduced by half at OHMXC concentrations < 20 LM.

































0 20 50 '100 200 300
IgM OHMXC


Figure 3-5. Autoradiogram showing the reverse-phase TLC separation of sulfonation
products of OHMXC.

Incubations were carried out with the indicated concentrations of OHMXC.
The arrow indicates the sulfate conjugate of the OHMXC, while other bands
represent unidentified sulfate conjugates formed from endobiotics or other
xenobiotics in polar bear liver cytosol.


The total TCPM sulfate conjugate production formed after 5 minutes under initial

rate conditions did not exceed 30 pmol. TLC, followed by autoradiography, was thus

used to distinguish the TCPM-sulfate band (Rf 0.54) from other sulfate conjugates (Rf

0.05 and 0.72) originating from compounds in the polar bear liver cytosol (Figure 3-6).

The data obtained followed hyperbolic kinetics (Table 3-2). Even though the TLC

from the kinetic experiments showed a TCPM concentration-dependent increase of the

band corresponding to the purported TCPM-sulfate, and this band was absent in the









substrate blank, the fact remained that we were apparently looking at the only instance

ever reported of a successful sulfonation of an acyclic tertiary alcohol.








*#4@










PC P100 CO C100 HO H100


Figure 3-6. Autoradiogram showing the reverse-phase TLC separation of sulfonation
products from incubations with TCPM using polar bear (P), channel catfish
(C), and human (H) liver cytosol in the absence of (0), and presence of 100
CLM TCPM (100).

The arrow indicates the sulfate conjugate of the substrate, while other bands
represent unidentified sulfate conjugates formed from endobiotics or other
xenobiotics in liver cytosol.


Thus, additional experiments were performed to verify the identity of this

conjugate. The purity of the TCPM was tested in the event that the additional band was

due to an impurity in the substrate. However, the substrate used was found to be free of

contaminants by HPLC (C18 reverse phase column, with detection at 268 and 220 nm,

using 90% methanol in water and a flow rate of 1 mL/min). A single peak was recorded









at 7.3 minutes. Another experiment involved a 60-minute incubation performed with 100

CLM TCPM and 0.1 mg cytosolic protein from polar bear, channel catfish and human

liver. For each of the three species, we detected a conjugate at Rf = 0.54. The substrate

blanks showed no band at the same position (Figure 3-6). The TCPM sulfate conjugate

from polar bear could be hydrolyzed by sulfatase (Figure 3-7), providing further evidence

of the sulfonation of this alcohol.

A B
12 34
















Figure 3-7. Autoradiogram showing the reverse-phase TLC separation of sulfonation
products of TCPM and the effect of sulfatase treatment.

A, incubation in the absence of TCPM (lane 1), and following treatment with
sulfatase (lane 2). B, incubation with 200 CLM TCPM (lane 3), and following
treatment with sulfatase (lane 4). The arrow indicates the sulfate conjugate of
the TCPM, while other bands represent unidentified sulfate conjugates formed
from endobiotics or other xenobiotics in polar bear liver cytosol.


Inhibition of sulfonation of substrates already present in the polar bear liver was

noted upon adding 1 CLM PCP (Figure 3-8). The data for PCP sulfonation fitted the

nonlinear Hill plot (eq. 2) (Table 3-2).






























0 1 2 5 10 20 50 75 100
E-M PCP


Figure 3-8. Autoradiogram showing reverse-phase TLC separation of sulfonation
products from the study of PCP kinetics.

The arrow indicates the sulfate conjugate of PCP, while other bands represent
unidentified sulfate conjugates formed from endobiotics or other xenobiotics
in polar bear liver cytosol.


Discussion

The sulfonation of hydroxylated metabolites of benzo[a]pyrene has been reported

in various species, including fish (James et al., 2001) and humans (Wang et al., 2004).

Benzo[a]pyrene-3 -glucuronide has been shown to be produced by fish (James et al.,

1997), rats (Lilienblum et al., 1987) and humans (Wang et al., 2004). There are,

however, few studies investigating the kinetics of these conjugation reactions.

Glucuronidation of 3-OH-B[a]P was more efficient in polar bear liver than in human liver

or catfish intestine. On the other hand, the efficiency of sulfonation was similar to that

shown in human liver but around three times less than in catfish intestine (Wang et al.,










2004, James et al., 2001). From the limited comparative data available, it can be surmised

that, in general, polar bear liver is an important site of 3-OH-B[a]P detoxication,

particularly with respect to glucuronidation.

Substrate inhibition for the sulfonation of 3-OH-B[a]P has been observed at

relatively low concentrations of the xenobiotic in other species such as catfish and human

(Tong and James 2000, Wang et al., 2005). Data from the polar bear sulfonation assay

fitted a two-substrate model developed for the sulfonation of 17P-estradiol by SULTIE

(Zhang et al., 1998). This model was also used to explain the sulfonation profie observed

for the biotransformation of 1-hydroxypyrene, a compound structurally similar to 3-OH-

B[a]P, by SULTs 1Al and 1A3 (Ma et al., 2003). In the original model, SULTIE1 was

saturated with PAPS, and each of the estradiol substrate molecules bound independently

to the enzyme. The estradiol binding sites were proposed to consist of a catalytic site, and

an allosteric site that regulates turnover of the substrate (Zhang et al., 1998). The

substrate inhibition observed with polar bear liver cytosol at higher 3-OH-B[a]P

concentrations (>0.75 pLM) can thus be explained by the binding of a second substrate

molecule to an allosteric site, which leads to a two-fold decrease in affinity and an

eightfold decrease in V;;;a.

SULTs are generally high-affinity, low-capacity biotransformation enzymes that

operate effectively at low substrate concentrations. Thus, typical K;;s for the sulfonation

of xenobiotic substrates are usually significantly lower than K;;s for the same substrates

undergoing biotransformation by low-affinity, high-capacity glucuronosyltransferases

(UGTs). In polar bear liver, both pathways showed similar apparent affinities for 3-OH-

B[a]P, with K;;s of 0.4 and 1.4 CLM for sulfonation and glucuronidation respectively,










suggesting these two pathways of Phase II metabolism compete at similar 3-OH-B[a]P

concentrations. However, the apparent maximal rate of sulfonation was about 7.5 times

lower than the rate of glucuronidation.

It was previously reported that the maximum rate of glucuronidation of 3-OH-

B[a]P by polar bear liver was 1.26 nmol/min/mg, or around half the V;;;a value obtained

in this study (Sacco and James 2004). However, the preceding study utilized 0.2 mM

UJDPGA, which, as seen from Table 3-2a, is equivalent to the K,; (for UDPGA) of the

low-affinity enzyme, and thus does not represent saturating concentrations of the co-

substrate. The affinity of the enzyme for 3-OH-B[a]P did not change significantly with a

20-fold increase in UDPGA concentrations, suggesting that substrate binding is

independent of the binding of co-substrate. The binding of UDPGA was biphasic,

indicating that multiple hepatic UGTs may be responsible for the biotransformation.

Biphasic UDPGA kinetics have also been demonstrated in human liver and kidney for 1-

naphthol, morphine, and 4-methylumbelliferone (Miners et al., 1988a,b; Tsoutsikos et al.,

2004). While V;;;a was similar for both components, there was a fivefold decrease in

enzyme affinity for UDPGA as the co-substrate concentration was increased. The

involvement of at least two enzymes can be physiologically advantageous since it enables

the maintenance of a high turnover rate even as UDPGA is consumed. Although

physiological UDPGA concentrations in polar bear liver are unknown, mammalian

hepatic UDPGA has been determined to be around 200-400 CLM (Zhivkov et al., 1975,

Cappiello et al., 1991), implying that the observed nonlinear kinetics in the polar bear

may operate in vivo.










The rate of triclosan sulfonation was the highest of all the substrates studied;

apparent V;;; was twice as high as for 3-OH-B[a]P. However, the overall efficiency of

sulfonation of the hydroxylated PAH was still 13 times higher than for triclosan

sulfonation. The presence of three chlorine substituents (though none flanking the phenol

group) does not hinder the sulfonation of triclosan when compared to the 'chlorine-free'

3-OH-B[a]P. Triclosan sulfonation in polar bear liver was similar to human liver with

respect to enzyme affinity; however the maximum rate was tenfold higher in polar bears

than in humans (Wang et al., 2004). This may be one reason why triclosan has not been

detected in polar bear plasma or liver to date.

Our data fitted a model that indicates the substrate inhibition observed for 4'-OH-

PCB79 may be due to a second substrate molecule interacting with the enzyme-substrate

complex at the active site rather than an allosteric site, resulting in a dead-end complex.

Unlike 3-OH-B[a]P, sulfonation can only proceed via the single substrate-SULT

complex. Models of SULTIAl and 1A3, with two molecules of p-nitrophenol or

dopamine at the active site respectively, have been proposed as a mechanism of substrate

inhibition (Gamage et al., 2003, Barnett et al., 2004), while the crystal structure of human

EST containing bound 4,4'-OH-3,3',5,5 '-tetrachlorobiphenyl at the active site has not

provided any evidence of an allosteric site (Shevtsov et al., 2003). The slower sulfonation

of 4'-OH-PCB79 compared with 3-OH-B[a]P may result from the inductive effect of the

chlorines flanking the phenolic group rather than steric hindrance (Duffel and Jakoby,

1981). However, polar bear liver sulfonated 4'-OH-PCB79 more rapidly than the other

OH-PCB substrates studied.









The inclusion of two additional chlorine substituents on the non-phenol ring (with

respect to 4'-OH-PCB79) resulted in both 4'-OH-PCBl59 and 4'-OH-PCBl65 being

very poor substrates. Ineffieient sulfonation may be one reason why the related

compound 4'-OH-PCBl72 accumulates in polar bears. Some degree of substrate

inhibition may also be expected to contribute to this accumulation, as was observed with

4'-OH-PCBl165.

Sulfonation was not an efficient pathway of OHMXC detoxification. The rate of

OHMXC-sulfonate formation was around 7 times lower than for 4'-OH-PCB79. Since

resonance delocalization of negative charge on the phenolic oxygen by the flanking

chlorines in chlorophenols may decrease Vmax by increasing the energy of the transition

state of the reaction (Duffel and Jakoby, 1981), it is possible that in the case of OHMXC

(with no chlorines flanking the phenolic group), product release, rather than sulfonate

transfer, may have been the rate-limiting step.

TCPM was a poor substrate for sulfonation, and this may be one reason why it has

been measured in such high amounts in polar bear liver. To our knowledge, sulfonation

of acyclic tertiary alcohols has not been reported in the literature. Despite the

considerable steric hindrance of three phenyl groups, the alcohol group could be

sulfonated. Although the alcohol in TCPM is not of the benzylic type, the presence of

three proximal phenyl groups may give this group some benzylic character, rendering

sulfonation of the alcohol possible. Both SULT 1El and SULT 2Al have been shown to

sulfonate benzylic alcohol groups attached to large molecules (Glatt, 2000). Sulfation of

the benzylic hydroxyl group leads to an unstable sulfate conjugate that readily degrades

to the reactive carbocation or spontaneously hydrolyzes back to the alcohol. Attempts to









recover TCPM-O-sulfonate from TLC plates resulted in recovery of TCPM from the

conjugate band, perhaps because of the conjugate' s instability.

A study of the sulfonation of PCP was complicated by the fact that it is a known

SULT inhibitor, often with K,s in the submicromolar range. In our experiments, this was

seen as a 74% decrease in formation of the unidentified sulfonate conjugates (band

shown at the solvent front in Figure 3-8) upon addition of 1 CLM PCP. Although PCP was

a strong inhibitor of SULTI1E1 (Kester et al., 2000), and has been postulated to be a dead-

end inhibitor for phenol sulfotransferases (Duffel and Jakoby, 1981), it was possible that

polar bear SULT 1A isoforms were not completely inhibited by PCP, or that other SULT

isoform(s) were responsible for the limited sulfonation activity observed. Thus, we have

shown that, in vitro at least, one mammalian species is capable of limited PCP

sulfonation. Even though the tertiary alcohol of TCPM was a poor candidate for

sulfonation, it was metabolized at twice the efficiency of PCP, which has a phenolic

group that is usually more susceptible to sulfonation. This demonstrates the extent of the

decreased nucleophilicity on the phenolic oxygen due to the resonance delocalization

afforded by the five chlorine substituents.

Conclusions

In summary, this study demonstrated that, in polar bear liver, 3-OH-B[a]P was a

good substrate for sulfonation and glucuronidation. Other, chlorinated, substrates were

biotransformed with less efficiency, implying that reduced rates of sulfonation may

contribute to the persistence of compounds such as hexachlorinated OH-PCBs, TCPM

and PCP in polar bear tissues.















CHAPTER 4
GLUCURONIDATION OF POLYCHLORINATED BIPHENYLOLS BY CHANNEL
CATFISH LIVER AND INTESTINE

Polychlorinated biphenyls (PCBs) were extensively used as dielectrics in the mid-

twentieth century. Despite a ban on their use in the US, Europe and Japan since the mid

1970s, the chemical stability of PCBs has resulted in their persistence at all trophic levels

around the globe. Enzyme-mediated biotransformation is an important influence on PCB

persistence, and its significance in PCB toxicokinetics is dependent on congener structure

and the metabolic capacity of the organism.

Polychlorinated biphenylols (OH-PCBs) are products of CYP-dependent oxidation

of PCBs (James 2001). While OH-PCBs are more polar than their parent molecules, they

are still lipophilic enough to be orally absorbed, and distribute to several tissues (Sinjari

et al., 1998). Thus, not only have these compounds been detected in the plasma (which

represents recent dietary exposure, biotransformation, and remobilization into the

circulation) of a variety of animal species, such as polar bear (Sandau et al., 2004),

bowhead whale (Hoekstra et al., 2003), catfish (Li et al., 2003), and humans (Fangstroim

et al., 2002; Hovander et al., 2002), but also significantly, from a developmental

toxicology aspect, in fetuses and breast milk (Sandau et al., 2002; Guvenius et al., 2003).

OH-PCBs may contribute significantly to the recognized toxic effects of PCBs such

as endocrine disruption (Safe 2001; Shiraishi et al., 2003), tumor promotion (VondrBkek

et al., 2005) and neurological dysfunction (Sharma and Kodavanti 2002; Meerts et al.,

2004).









Elimination of these toxic metabolites via Phase II conjugation reactions, such as

glucuronidation and sulfonation, are thus important routes of detoxification. In view of

the persistence of certain OH-PCBs, it is surprising that only a few studies have

attempted to investigate the biotransformation of these compounds in animals or humans,

particularly by glucuronidation (Tampal et al., 2002; Sacco and James 2004; Daidoji et

al., 2005), which is normally a higher-capacity pathway than sulfonation.

Glucuronidation is catalyzed by a family of endoplasmic reticular membrane-bound

enzymes, the UDP-glucuronosyltransferases (UGTs), which transfer a D-glucuronic acid

moiety from the co-substrate UDP-glucuronic acid (UDPGA) to a xenobiotic containing

a suitable nucleophilic atom such as oxygen, nitrogen and sulfur. UGTs are mainly found

in the liver, but also in extrahepatic tissues, such as the small intestine and kidney (Wells

et al., 2004).

The various chlorine and hydroxyl substitution patterns possible on the biphenyl

structure may lead to significant differences in glucuronidation kinetics. One explanation

for the retention of certain OH-PCBs may thus be that they are poor substrates for

glucuronidation. Tampal and co-workers (2002) studied the glucuronidation of a series of

OH-PCBs by rat liver microsomes. Efficiency of glucuronidation varied widely, and

substitution of chlorine atoms at the m- and p-positions on the nonphenolic ring greatly

lowered Vmax. Weak relationships were observed between the dihedral angle, pKa, log D

and enzyme activity. The experimentally determined kinetic parameters determined in the

Tampal et al study were subsequently related to the physicochemical properties and

structural features of the OH-PCBs by means of a quantitative structure-activity









relationship (QSAR) study. Hydrophobic and electronic aspects of OH-PCBs were shown

to be important in their glucuronidation (Wang, 2005).

Most of the persistent OH-PCBs found in human plasma are hydroxylated at the p-

position, in addition to being meta-chlorinated on either side of the phenolic group. The

remaining substitution pattern on both rings is highly variable (Bergman et al., 1994;

Sjoidin et al., 2000). An OH group in the para position, with two flanking chlorine atoms

was associated with estrogen and thyroid hormone sulfotransferase inhibitory activity

(Kester et al., 2000; Schuur et al., 1998), and exhibited the highest affinity for

transthyretin (TTR) (Lans et al., 1993), the major transport protein in non-mammalian

species (Cheek et al., 1999). Such OH-PCBs were potent inhibitors of the sulfonation of

3 -hydroxybenzo[a]pyrene (Wang et al., 2005). In contrast, the OH-PCBs having an

unhindered hydroxyl group substituted at the para position (relative to the biphenyl bond)

have exhibited the strongest binding to the rodent estrogen receptor (ER), although the

competitive ER binding affinities were <100-fold lower than that observed for estradiol

(Korach et al., 1988; Arulmozhiraja et al., 2005).

In the channel catfish, individual OH-PCBs have been shown to inhibit the in vitro

intestinal glucuronidation of several hydroxylated metabolites of benzo[a]pyrene (BaP)

(van der Hurk et al., 2002; James and Rowland-Faux 2003). The in situ hepatic

glucuronidation of a procarcinogenic BaP metabolite, the (-)benzo[a]pyrene-7,8-dihydro-

diol, was also inhibited by a mixture of OH-PCBs, consequently increasing the formation

of DNA adducts (James et al., 2004). It is possible that these compounds inhibit their own

glucuronidation. The OH-PCB metabolites of 3,3,4,4-tetrachlorobiphenyl (CB-77), one

of the most toxic PCBs known, were poor substrates for catfish intestinal glucuronidation









(James and Rowland-Faux, 2003). This may help to explain the persistence of these

compounds.

Hypothesis

The glucuronidation kinetics of a series of potentially toxic p-OH-PCBs by channel

catfish liver and proximal intestine is influenced by the structural arrangement of the

chlorine substituents around the biphenyl ring.

Methodology

Chemicals. A total of 14 substrates were used in this study (Figure 4-1). The

nomenclature of the OH-PCBs is based on the recommendations of Maervoet and co-

workers (2004).

The following substrates (Catalog no. in parentheses) were purchased from

Accustandard (New Haven, CT): 4-OHCB2 (1003N), 4-OHCBl4 (2004N), 4'-OHCB69

(4008N), 4'-OHCB72 (4009N), 4'-OHCB106 (5005N), 4'-OHCBll2 (5006N), 4'-

OHCBl21 (5007N), 4'-OHCBl59 (6001N), and 4'-OHCBl65 (6002N). The compounds

4'-OHCB35, 4-OHCB39, 4'OHCB68, 4'-OHCB79 were synthesized by Suzuki-coupling

(Lehmler and Robertson, 2001; Bauer et al., 1995). The 4-hydroxy biphenyl (4-OHBP)

was purchased from Sigma (St.Louis, MO). 14C-UDPGA (196 CICi/Clmol) was obtained

from PerkinElmer Life and Analytical Sciences (Boston, MA). The 14C-UDPGA was

diluted with unlabelled UDPGA to a specific activity of 1.5-5 CICi/Clmol for use in

enzyme assays. PIC-A (tetrabutylammonium hydrogen sulfate) was obtained from

Waters Corp. (Milford, MA). Other reagents were the highest grade available from Fisher

Scientific (Atlanta, GA) and Sigma.


















4-OHCB2


4' -OHCB3 5


4-OHCB39 4' -OHCB6 8

CICl


Cl ClI Cl' Cl

OH
OH

4' -OHCB79 4' -OHCB 106


4' -OHCB72


4' -OHCB 112


Cl Cl
4' -OHCB l59 4' -OHCBl165


Figure 4-1. Structure of substrates used in channel catfish glucuronidation study.

Animals. Channel catfish (Ictalurus punctatus), with weights ranging from 2.1 -

3.7 kg, were used for this study. All fish were kept in flowing well water and fed a fish

chow diet (Silvercup, Murray, UT). Care and treatment of the animals was conducted as

per the guidelines of the University of Florida Institutional Animal Care and Use

Committee. The microsomal fractions were obtained from liver and intestinal mucosa


OH

4-OHCB14





-OH

4' -OHCB69


OH

4-OHBP


I~ OH

4' -OHCB l21










using a procedure described previously (James et al., 1997). Only the proximal portion of

the intestine was used in the study. Protein determination was carried out by the method

of Lowry and co-workers (195 1) using bovine serum albumin as protein standard.

Glucuronidation assay. A radiochemical ion-pair extraction method was

employed to investigate the glucuronidation of the 4-OHPCBs and 4-OHBP. Substrate

consumption did not exceed 10%. Initial experiments determined the saturating

concentrations of UDPGA to be employed. The incubation mixture consisted of 0.1 M

Tris-Cl buffer (pH 7.6), 5 mM MgCl2, 0.5% Brij-58, 200 CIM or 1500 CIM [14C]UDPGA

(intestine and liver, respectively), 100 Clg catfish intestinal or hepatic microsomal protein,

and substrate in a total reaction volume of 0. 1 mL. Initially, the OH-PCBs were added to

tubes from methanol solutions and evaporated under nitrogen. In all cases, the protein

and Brij-58 were added to the dried substrate, thoroughly vortexed and left on ice for 30

minutes. Subsequently, the buffer, MgCl2, and water were added in that order and vortex-

mixed. After a pre-incubation of 3 minutes at 35oC, UDPGA was added to initiate the

reaction, which was terminated after 30 minutes incubation by the addition of a 1:1

mixture of 2.5% acetic acid and PIC-A in water, such that the final volume was 0.5 mL.

The glucuronide product was extracted by two successive 1.5 mL portions of ethyl

acetate. The phases were separated by centrifugation, and duplicate portions of the ethyl

acetate phase were counted for quantitation of glucuronide conjugate.

Physicochemical parameters. The structural characteristics of the OH-PCBs were

calculated using ChemDraw 3D (CambridgeSoft Corp., Cambridge, MA). Parameters

used were: the Connolly Accessible Surface Area (CAA, the locus of the center of a

probe sphere, representing the solvent, as it is rolled over the molecular shape), the









Connolly Molecular Surface Area (CMA, the contact surface created when a probe

sphere (radius = 1.4 A+, the size of H20), representing the solvent, is rolled over the

molecular shape), the Connolly Solvent-Excluded Volume (CSV, the volume contained

within the contact molecular surface, or that volume of space that the probe is excluded

from by collisions with the atoms of the molecule), the ovality (the ratio of the Molecular

Surface Area to the Minimum Surface Area, which is the surface area of a sphere having

a volume equal to CSV of the molecule), and dihedral angle (the angle formed between

the planes of the two rings, which is related to the extent of coplanarity of the molecule).

ACD/ILab software (Advanced Chemistry Development, Ontario, Canada) was used to

predict log P, log D (at pH 7.0), and the pKa (of the phenolic group).

Kinetic analysis. Duplicate values were employed for the rate of conjugate

formation at each substrate concentration to calculate kinetic parameters using Prism v4.0

(GraphPad Software, Inc., San Diego, CA). Equations used to fit the data were the

Michaelis-Menten hyperbola for one-site binding and the Hill plot for positive

cooperativity.

Results

The kinetics for UDPGA were analyzed for the glucuronidation of three

representative OH-PCBs (Table 4-1). Saturating concentrations of UDPGA were higher

in liver than in intestine (Figure 4-2). The glucuronidation of most of the OH-PCBs tested

followed Michaelis-Menten kinetics (Figure 4-3A). In the case of the glucuronidation of

4'OHCB35 by liver and 4'OHCBll2 by proximal intestine, the data fitted the Hill plot

(Figure 4-3B).









Table 4-1. Estimated kinetic parameters (mean & S.D.) for the co-substrate UDPGA in
the glucuronidation of three different OH-PCBs.
Substrate Substrate Vmax (app) Km (app)
Concentration (CIM) (nmol/min/mg) (CLM)
Liver
4'-OHCB-3 5 500 0.87 & 0.20 697 & 246

4'-OHCB-72 250 0.32 & 0.14 247 & 162

Intestine
4'-OHCB-69 200 0.20 + 0. 11 27 & 14




The estimated apparent maximal rate of glucuronidation of polychlorinated

biphenylols by channel catfish ranged from 124-784 pmol/min/mg for proximal intestine

and 404-2838 pmol/min/mg for the liver (Table 4-2). The Kms for individual OH-PCBs

tended to be different in the two organs, with a few exceptions (40HCB2, 4'OHCBl65).

Vmax was significantly higher in liver than in intestine. Conversely, the affinity of

intestinal catfish UGTs (Km range: 42-572 C1M) for the OH-PCBs tested was higher than

for liver UGTs (Km range: 111-1643 CIM). These contrasting differences are reflected in

the lack of any difference in the efficiency of glucuronidation in both organs when all the

OH-PCB substrates were considered (Table 4-3). Vmax for OH-PCB glucuronidation in

both organs were strongly correlated with each other (R2=0.74). This relationship did not

exist for Km (R2=0.003).




























[UDPGA] (CIM)


0 25 50 75 100


[UDPGA] (CIM)


Figure 4-2. UDPGA glucuronidation kinetics in 4 catfish.

A) in liver, using 500 CLM 4'-OH-CB35. B) in proximal intestine, using 200
CLM 4'-OH CB69



























0 250 500 750 1000 1250 1500


[4'-OH CB 159] (CLM)


0 100 200 300 400 500 600 700


[4'-OH CB 112] (CIM)


Figure 4-3. Representative kinetics of the glucuronidation of OH-PCBs in 4 catfish.

A) Michaelis-Menten plot for 4'-OHCB-159 by liver. B) Hill plot for 4'-
OHCB-112 by proximal intestine





Units for Km and Vmax are CLM and pmol/min/mg protein, respectively. Bold indicates Sso
in place of Km. ND, not done.

Table 4-3. Comparison of the estimated kinetic parameters for OH-PCB glucuronidation
in catfish liver and proximal intestine
Parameter Liver Intestine p-value

Vmax (app) 1370 & 275 364 & 70 0.002

Km (app) 567 & 128 210 & 46 0.016

Vmax/Km 3.7 & 0.6 3.4 & 1.8 0.857

(Mean & SEM for all OH-PCB substrates)


Table 4-2. Kinetic parameters (Mean & S.D.) for the glucuronidation of 4-OHBP and OH-
PCBs.


Intestine
Substrate Vmax (app) Km (app)


Liver
Vmax (app)


Km (app)


4-OHBP

4-OHCB2

4-OHCB14

4'-OHCB3 5

4-OHCB 3 9

4'-OHCB68

4'-OHCB69

4'-OHCB72

4'-OHCB79

4'-OHCB 106

4'-OHCBl12

4'-OHCBl121

4'-OHCBl159

4'-OHCBl165


43 A 10

417 & 57

255 & 59

784 & 348

220 + 90

213 & 91

751 & 253

401 & 236

124 & 36

431 & 60

401 & 67

220 & 39

188 & 66

163 & 26


599 & 110

572 & 47

387 & 65

265 & 85

134 & 36

119 & 75

42 & 21

183 A 126

87 & 21

183 & 58

163 & 24

130 & 21

213 A 136

137 & 44


182 & 78

2277 & 849

2022 & 936

2838 & 1456

1716 & 536

ND

2774 & 1153

ND

869 & 318

1579 & 645

2144 & 1007

1046 & 408

681 +141

404 & 116


502 & 235

583 & 95

614 & 202

455 & 89

242 & 76

ND

1071 & 410

ND

476 & 201

798 & 122

1643 & 545

207 & 97

318 & 91

111 & 28









The Vmax for glucuronidation in both proximal intestine and liver was significantly

decreased upon addition of a second chlorine substituent flanking the phenolic moiety,

while keeping the chlorine substitution pattern in the rest of the molecule constant (Table

4-4, Figure 4-4). The affinity of hepatic UGTs for the OH-PCBs appeared to increase

with the addition of a second flanking chlorine atom; however, this relationship did not

achieve statistical significance.


Table 4-4. Comparison of kinetic parameters (Mean & SEM) for the glucuronidation of
OH- PCBs grouped according to the number of chlorine atoms flanking the
phenolic group

Parameter Flanking chlorines p-value
1 2


Liver

Vmax (app), pmol/min/mg 2247 & 204 1002 & 274 0.007

Km (app), CIM 856 & 209 342 & 88 0.053


Intestine

Vmax (app), pmol/min/mg 560 + 85 190 & 23 0.003
Km (app), CIM 274 & 97 191 & 53 0.473




The effect of chlorine substituents on the nonphenolic ring on glucuronidation of

OH-PCBs was also investigated. No significant differences on Km and Vmax could be

observed between the absence or presence of specific chlorine substituents on the

nonphenolic ring. The only exception was that the presence of an ortho-chlorine

significantly (p=0.03) decreased the Km in the proximal intestine.














































U


I one flanking Cl
EC: two flanking Cl


0 3,4 2,4,6 2,3,4,5 2,3,5,6
Substitution pattern on nonphenol ring


Sone flanking Cl
..I~ two flanking Cl


40001

30001

2000-

1000-


~I


03,4 2,4,6
Substitution pattern on


2,3,4,5 2,3,5,6
nonphenol ring


Figure 4-4. Decrease in Vmax with addition of second chlorine atom flanking the phenolic
group, while keeping the chlorine substitution pattern on the nonphenolic ring
constant.


A) proximal intestine. B) liver.


I










Regression analysis was performed between the kinetic parameters for the

glucuronidation of OH-PCBs and several physical parameters for these substrates (Table

4-5). The data for 40HBP was not used since this compound is not a OH-PCB. The

affinity of intestinal UGTs was negatively correlated with the Connolly solvent-

accessible surface area, the molecular surface area, solvent-excluded volume, ovality,

dihedral angle, log P, and positively correlated with pKa. The maximum rate of hepatic

glucuronidation was negatively correlated with the Connolly solvent-accessible surface

area, the molecular surface area, solvent-excluded volume, ovality, and log P, and

positively correlated with pKa (which showed a similar relationship with intestinal Vmax).

Ovality was also significantly negatively correlated with the maximum rate of intestinal

glucuronidation of the OH-PCBs studied (Figure 4-5).

A paired t-test performed in order to investigate the physicochemical parameters

involved in the significant decrease in Vmax observed for the glucuronidation of OH-

PCBs with two chlorine atoms flanking the phenolic group revealed that, for OH-PCBs

with this structural arrangement, pKa was decreased (p=0.02), while log P, and

parameters indicating molecular size (CAA, CMA, CSEV, ovality) were all increased (all

p <0.0001).




















CAA




CMA




CSEV




Ovality




Dihedral

angle


log P




log D
(pH 7.0)


pKa


Sign in parentheses indicates type of correlation where it achieved significance.


Physical
Parameter


Table 4-5. Results of regression analysis performed in order to investigate the
relationship between the glucuronidation of OH-PCBs by catfish proximal
intestine and liver and various estimated physical parameters.


Intestine

Vmax (app)


0.060
0.079


0.056
0.087


0.047
0.118




0.014


0.002
0.755


0.058
0.086


0.011
0.467


0.143 (+)
0.006


Liver

Vmax (app)


0.253 (-)
0.0005


0.249 (-)
0.0006


0.236 (-)
0.0008


0.286 (-)
0.0002


0.077
0.068


0.250 (-)
0.003


0.015
0.490


0.306 (+)
0.0007


Statistic


R2

p-value


R2

p-value


R2

p-value


R2

p-value


R2

p-value


R2

p-value


R2

p-value


R2

p-value


Km (app)


0.439 (-)
<0.0001


0.423 (-)
<0.0001


0.396 (-)
<0.0001


0.431 (-)
<0.0001


0.248 (-)
0.0002




0.044


0.035
0.271


0.108 (+)
0.047


Km (app)


0.004
0.685


0.002
0.780


<0.001
0.962


0.068
0.088


0.026
0.296


0.061
0.137


<0.001
0.963


0.093
0.063











'-- a Vmax (int)
g a Vmax (liv)









1.360 1.385 1.410 1.435
ovality




Figure 4-5. Relationship between Vmax for OH-PCB glucuronidation in intestine and liver
and ovality

Discussion

In comparison to catfish intestine, catfish liver displayed higher rates of

glucuronidation of OH-PCBs, however both organs collectively biotransform the OH-

PCBs studied with similar efficiency. This occurred because while the glucuronidation

Vmax in the intestine was lower than in the liver, the affinity of intestinal UGTs for the

OH-PCBs was higher than liver UGTs. However, the efficiency of glucuronidation of 4'-

OHCB69 was seven times higher in the proximal intestine; when the data for this

substrate was excluded, the efficiency of glucuronidation was significantly higher

(p=0.01) in liver.

The total UGT capacity in the liver is much greater than in intestine when the total

content of microsomal protein in these two organs is taken into consideration. In fact, the

levels of microsomal protein from liver were always higher than in the intestine of each

individual fish studied, possibly because of the decreased amount of endoplasmic









reticulum in enterocytes relative to hepatocytes (DePierre et al., 1987). Thus, the intestine

appears to compensate for the lower glucuronidation capacity by expressing UGTs with a

higher affinity.

No relationship was established between Kms for the glucuronidation of OH-PCBs

in liver and intestine. When individual OH-PCBs were considered, there were significant

differences in efficiency. These results suggest that these two organs have different UGT

isoform profiles, with the intestine possessing one or more isoforms that display greater

specificity for OH-PCBs. Possible UGT isoforms responsible may be catfish enzymes

analogous to rat UGTIA1, UGTIA6 and UGT2B1 (Daidoji et al., 2005), and to plaice

hepatic UGTIBl, which has been shown to conjugate planar phenols (Clarke et al.,

1992).

The substrates 4'-OHCB69 and 4-OHCB39 were glucuronidated with the highest

efficiency in the intestine and liver respectively. 4'-OHCB35 showed the highest rates of

glucuronidation in both liver and intestine. The poorest substrates were 4-OHCBl4 in the

intestine and 4'-OHCBll2 in the liver. In contrast, rat liver glucuronidates 4-OHCBl4

with the highest efficiency, relative to other OH-PCBs studied (Tampal et al., 2002).

Overall, the efficiency of glucuronidation of the OH-PCBs by rat liver is higher than in

catfish liver. While these dissimilarities may be ascribed to differences in UGT isoform

type and expression due to the different species and tissues in the two studies, it may also

indicate an increased susceptibility of catfish to the toxic effects of OH-PCBs due to an

increased bioavailability.

Compared to the OH-PCBs, 4-OHBP was the poorest substrate for glucuronidation.

This compound had the lowest Vmax in both liver and proximal intestine. The affinity for









4-OHBP in the intestine was also the lowest. In the liver however, the Km was

comparable to other OH-PCBs. These results are surprising in view of the fact that 4-

OHBP has been shown to be a good substrate for glucuronidation using rat, guinea pig,

beagle dog and rhesus monkey liver microsomes (Yoshimura et al., 1992), and human

expressed UGTs (King et al., 2000; Ethell et al., 2002). In isolated rat hepatocytes, 4-

OHBP is a cytotoxic major metabolite of biphenyl, impairing oxidative phosphorylation

(Nakagawa et al., 1993). These results suggest that this compound may be potentially

more toxic to catfish than to mammals, unless cleared by another pathway such as

sulfonation.

While the decreased glucuronidation of 4-OHBP may be due to the lack of a

specific phenol UGT isoform in catfish, the known broad substrate specificity of phenol

UGTs, together with the observed higher rates of glucuronidation for the OH-PCBs, leads

us to hypothesize that this compound may be such a poor substrate due to its lower

lipophilicity, as has been observed for other substituted phenols (Kim 1991). In fact,

addition of a single chlorine atom flanking the phenolic group (as represented by

40HCB2) resulted in at least a tenfold increase in Vmax in both liver and intestine, with no

significant change in Km (with respect to 4-OHBP). This increased lipophilicity

(represented by an estimated log P increase from 3.2 to 3.8) appeared to impact the

formation of the glucuronide and not the initial binding of substrate to UGT. Good UGT

substrates tend to be lipophilic compounds which are thought to diffuse through the

endoplasmic reticular bilayer and reach the substrate-binding site in the lumenal N-

terminal part of the enzyme, which contains a region of strong interaction with the

membrane (Radominska-Pandya et al., 2005). For all the OH-PCBs studied, we only









observed weak inverse correlations (R2<0.3) between log P and intestinal Km and liver

Vmax. No significant relationship could be observed between parameters of lipophilicity

and intestinal Vmax. The absence and weakness of such relationships may reflect the need

for OH-PCBs with additional structural variation to be included in studies of this type.

Another explanation may be the perturbation of the lipid bilayer of the microsomes,

resulting in rate-limiting partitioning, which would not be present in vivo (Tampal et al.,

2002).

As the estimated pKa of the OH-PCBs increased, so did hepatic and intestinal Vmax

for glucuronidation. These results are in agreement with a previous OH-PCB

glucuronidation study in rats (Tampal et al., 2002). Thus, a greater proportion of ionized

OH-PCB molecules appear to have an adverse effect on glucuronidation. Such charged

molecules present at the active site of UGT may interfere with the charge-relay system

that relies on a basic negatively charged residue to deprotonate the phenolic group, prior

to transfer of glucuronic acid (Yin et al., 1994).

Since the use of microsomal systems to elucidate structure-activity relationships

involves incubations of substrate with a heterogeneous population of UGTs exhibiting

different levels of expression and activity, it was not the intention of this study to attempt

to predict the effect of molecular structure and physicochemical parameters on the

glucuronidation of OH-PCBs, which is better achieved using individual isoforms.

However, if any such effects can be observed at a microsomal level, then it is likely that

such processes are occurring in the organism, whose detoxification route depends on

various UGTs metabolizing substrate simultaneously and not in isolation. This may help

to further delineate the different toxicokinetics of OH-PCBs.









The p-OH-PCBs used in this study all had one or two chlorine atoms flanking the

phenolic group. This structural motif is of interest since it imparts several toxic properties

to these compounds. OH-PCBs with two flanking chlorines were found to be poorer

substrates than compounds with one flanking chlorine atom, in both liver and intestine.

Thus, for example, while 4'-OHCB35 was a very good substrate for glucuronidation,

addition of a second flanking chlorine (as in 4'-OHCB79) resulted in a greater decrease in

Vmax than the addition of two adjacent chlorine substituents on the aphenolic ring (as in

4'-OHCB 106). A comparison of the physicochemical parameters of the two different

structural arrangements suggests that lipophilicity, pKa, and molecular size may all be

contributing to this effect on Vmax.

The addition of a second chlorine atom imparts additional lipophilicity to the

molecule and may increase positive charge on the phenolic carbon atom, which results in

stronger binding to the active site (Wang 2005). This study did show a non-significant

decrease in Km with the addition of the second chlorine atom for both organs. On the

other hand, the 3,5-chlorine substitution pattern may interfere with the mechanism of

glucuronidation because of steric hindrance, although this has been disputed (Mulder and

Van Doorn 1975; Tampal et al., 2002).

The estimated pKas for OH-PCBs with two flanking chlorine substituents were

significantly lower than similar molecules with one flanking chlorine atom. This is

supported by limited experimental data showing that OH-PCBs with two flanking

chlorine atoms have pKa values as low as 6.4 (for 4'-OHCB39, Miller 1978). The

population of OH-PCB molecules which are ionized at physiological pH is significantly

more than OH-PCBs with one flanking chlorine atom, resulting in the adverse effect on









the enzymatically-catalyzed charged relay system described above. In studies conducted

with rat liver microsomes, a decreased maximal rate of glucuronidation was also

observed amongst OH-PCBs differing only in the number of chlorines flanking the

phenolic group (1 pair of OH-PCBs in Tampal et al., 2002; 2 pairs of OH-PCBs in

Daidoji et al., 2005). According to Daidoji and co-workers (2005), UGT2B1 is the

primary rat hepatic UGT isoform responsible for metabolizing OH-PCBs with one

flanking chlorine atom. UGTIAl appears to metabolize both, though with a preference

for structures with two flanking chlorines

These results are significant from a toxicological standpoint since almost all the

major OH-PCBs found in human plasma incorporate a 4'-hydroxy-3',5' -dichloro

structure (Sandau et al., 2002; Fangstrom et al., 2002; Hovander et al., 2002). It is

possible that one reason for the persistence of these OH-PCBs may be a reduced rate of

glucuronidation due to this structural arrangement.

Two or more chlorine substituents that are ortho to the biphenyl bond cause the

molecule to twist and assume a non-coplanar conformation. In the parent PCBs this leads

to toxicological differences, such as loss of AhR agonist activity. The estimated dihedral

angles for the compounds investigated in this study ranged from 360-760. The affinity of

intestinal, but not hepatic, UGTs appeared to increase with the degree of twisting,

suggesting that the predominant isoform(s) in catfish intestine binds more strongly to the

more twisted OH-PCBs. While this may be additional evidence of differences with

respect to isoform profiles between liver and intestine, the weakness of the relationship

(R2~0.3) precludes using this result to solidly support this hypothesis.









Similar to what has been reported for the glucuronidation of OH-PCBs in rats

(Tampal et al., 2002) and simple phenols by human UGTIA6 (Ethell et al., 2002), the

maximal rate of hepatic glucuronidation decreased with increased steric bulk. In the case

of intestinal glucuronidation this relationship was weaker. The enzyme affinity of

intestinal UGTs increased with increasing molecular size, perhaps because the bulkier

molecules tended to be more lipophilic. However, in contrast, the affinity of the liver

UGTs was not affected as much by the molecular size, at least within the restricted size

range offered by the OH-PCBs studied. At this point, no explanation for this discrepancy

between these two tissues is forthcoming.

Conclusions and Recommendations

OH-PCBs are glucuronidated with similar efficiency by channel catfish liver and

proximal intestine. There appear to be differences in the UGT isozyme profile in both

organs. The Vmax for both hepatic and intestinal glucuronidation was decreased with the

addition of a second chlorine atom flanking the phenolic group, which is an arrangement

typical of OH-PCBs that persist in organisms. Future research may be directed towards

cloning, sequencing and characterizing these catfish UGTs, in order to have a better

understanding of the specificity of individual UGT isoforms for particular chlorine

substitution patterns in OH-PCBs.















CHAPTER 5
CLONING OF UDP-GLUCURONOSYLTRANSFERASES FROM CHANNEL
CATFISH LIVER AND INTESTINE

Piscine UGT Gene Structure and Isoforms

Fish are the most ancient vertebrate phylum, and account for over 40% of all living

vertebrate species (Clarke et al. 1992a). Clarke and co-workers (1992b) compared the

hepatic glucuronidation of several xenobiotics and endobiotics in plaice (Pleuronectes

platessa) and rat (Rattus norvegicus), species that are separated by more than 350 million

years of evolutionary divergence. Despite the fact that the plaice showed reduced

glucuronidation activity towards substrates such as morphine, bilirubin and steroids,

weak immunological cross-reactivity was obtained when anti-rat UGT antibodies were

used, indicating the presence of conserved common structural motifs between the two

vertebrates.

Characterization of plaice UGTIB 1 (Accession number (AN): X741 16), an isoform

which conjugates planar phenols and is inducible by polyaromatic hydrocarbons (PAH),

confirmed the strong degree of conservation in gross exon structure and amino acid

character (signal peptide, membrane insertion, and stop sequences) between fish and

mammals. The greatest degree of similarity in amino acid sequence was found with

UGT1 rather than UGT2 (Clarke et al., 1992b, George et al., 1998). Allelic variations in

this UGTIB1 gene are presumed to be functionally silent (George and Leaver 2002).

While there is strong evidence for other distinct isoforms conjugating bilirubin, estrogen

and androgens, to date these have not been characterized. At least six distinct UGTs









exhibited tissue-specific expression in plaice (Clarke et al., 1992c). UGTIB2 mRNA has

recently been sequenced from marbled sole (Pleuronectes yokohamnae) liver (AN:

ABl20133), and a partial sequence of an unidentified UGT isoform has been obtained

from the orange-spotted grouper (Epinephehis coioides) (AN: AY735003). The existence

of a number of partial length sequences of UGT homologues from zebrafish (Dario rerio)

EST projects in GenBank provide evidence for the cDNA of 10 distinct UGTs. The

absence of cDNAs with the same 3'sequence and dissimilar 5-exon 1 coding sequence

suggests the absence of alternative splicing of UGTIA genes as seen in mammals. Thus,

George and Taylor (2002) have suggested the existence of three family 1-related UGTs

and another two related to the UGT 2 family in the zebrafish. In general, however, it

appears that fish possess multiple UGTs with similar functional and structural properties

to mammalian UGT.

Toxicologically, it is important to know whether xenobiotic pollutants such as

PAHs compete with steroids or bilirubin for the same active site on UGT, resulting in

physiological perturbations in reproductive and/or liver function. For example, Atlantic

salmon (Salmo salar) suffering from a multiple pollutant-induced j aundice were shown to

have decreased bilirubin UGT activity (George et al. 1992). Channel catfish are also

exposed to pollutants (such as PAHs and PCBs) which accumulate in sediments. Thus,

this organism may be a useful indicator of the bioavailability of these pollutants in such

sedimentary environments. In addition, the use of this Hish in aquaculture makes it

essential to understand every aspect of its detoxification mechanisms, since these will

ultimately impact human health.









While no UGTs have yet been cloned and characterized from channel catfish, this

species shows glucuronidation activity towards a variety of toxic xenobiotics, including

mono- and di-hydroxy metabolites of benzo[a]pyrene and OH-PCBs (James et al., 2001;

van den Hurk and James 2001; Gaworecki et al., 2004). As with other aquatic species,

pollutants which are direct substrates for glucuronidation, such as pentachlorophenol,

several OH-PCBs, 4-OH-heptachlorostyrene, and which have been shown to be

estrogenic and thyroidogenic, have been detected in channel catfish (Li et al., 2003).

Kinetic differences have been observed between hepatic and intestinal UGT activities,

suggesting expression of different isozymes in these two organs. Thus, knowing more

about the identity and substrate specificity of catfish UGTs will assist our understanding

of the effect of glucuronidation on the contributions of such metabolites to toxicity. Since

the absence of cDNAs with the same 3'sequence and dissimilar 5'exon 1 coding

sequence in fish suggests the absence of alternative splicing of UGTIA genes as seen in

mammals (Gong et al., 2001), additional information on piscine UGT gene structure is

also important from a phylogenetic perspective.

Hypothesis

Multiple UGT isoforms are present in channel catfish liver and intestine

Methodology (part 1)

For convenience, a flowchart summarizing the various steps involved in the cloning

process is shown in Figure 5-1. Because the study utilizing the gene specific primers was

dependent on an initial study which utilized degenerate primers and led to the cloning of

partial sequences of UGT, the methodology and results sections are split correspondingly

mn two parts.


























J


. _ _


Sequencing BLAST search
of partial
1. Des n GS primers
length UGTs 2. 5' and 3' RLMf-RACE
3. Clone and sequence

Sequence overlap
1. Des n GS primers
2. PCR with Super Taq Plus
3. Clone and sequence

Full-length cDNA clone


Figure 5-1. Summary of methods used to clone channel catfish UGT

Animals. A single female adult catfish was sacrificed. Total weights of liver and

intestinal mucosa were recorded. Tissues were immediately processed for RNA isolation.

RNA isolation. Approximately 0.1Ig of tissue from the liver and proximal intestinal

mucosa were homogenized in separate tubes with 1 mL Trizol@ reagent and placed on

ice. The homogenates were incubated for 5 min at room temperature (15-300C) to enable

complete dissociation of nucleoprotein complexes. Chloroform, 0.2 mL, was added and


catfish liver/intestine

1. RNA isolation
2. RT-PCR, using degenerate primers based on consensus sequences


U GT / ~a ion
cDNA f O transformation

pGEM T-Easy vector recombinant plasmid

bacterial/lasmid
replica tion
plasmid
purification!
UGT clones Ecoi ,MO









the tubes were shaken vigorously by hand for 15 seconds and then incubated at room

temperature for 2-3 min. The samples were then centrifuged at 12,000g for 15 min at 2-

80C. This separated the solution into an aqueous phase containing the RNA and an

organic phase containing DNA. The colorless upper aqueous phase was transferred to an

RNase-free tube. The RNA was precipitated by the addition of 0.5 mL propan-2-ol. The

samples were then incubated at room temperature for 10 min, followed by centrifugation

at 12,000g for 10 min at 2-80C. The RNA precipitate was now visible as a gel-like pellet

on the side and bottom of the tube. The supernatant was removed and the RNA pellet was

washed once with 1 mL 75% ethanol. The sample was vortex-mixed and centrifuged at

7,500g for 5 minutes at 2-80C. The RNA pellet was left to air-dry for a few minutes

following decantation of the ethanol. The RNA was dissolved in 100 CIL RNase-free

water for intestine, and 200 CIL RNase-free water for liver (since the solution in this case

appeared to be more concentrated), by passing the solution a few times through a pipette

tip. The solution was then incubated for 10 minutes at 55-600C. The samples were stored

at -800C. The purity of the RNA was checked by running the sample on 1% agarose gel

(with 9.5% formaldehyde) and 10x MOPS buffer. Bands corresponding to the 28S and

18S ribosomal subunits were observed. The purity of the RNA was also checked by

diluting the sample in 10mM Tris HC1, pH 7.5 and measuring the A260/A280 absorbance

ratio (ideally should be between 1.8 and 2.1i).

DNase treatment of RNA samples. This procedure was done in order to remove

contaminating DNA from RNA preparations, and to subsequently remove the DNase and

divalent cations from the sample. Portions of the RNA solutions were diluted to 100

Clg/mL with RNase-free water. The Ambion 9 (Austin, TX) DNA-removal kit was used.










The reaction mix, consisting of 25 CIL RNA, 2.5 CIL 10xDNasel buffer, and 3 CIL DNase I

was incubated at 370C for 1 hour. DNase inactivation reagent, 5 CIL, was added by means

of a wide pipette tip (due to the thick consistency of this reagent). The tubes were then

incubated for 2 min at room temperature, with gentle flicking. The tubes were then

centrifuged at 10,000g for ~1 min to pellet the DNase inactivation reagent. The

supernatant containing the RNA was transferred to a new RNase-free tube and stored at -

800C.

Generation of cDNA library. The Retroscript@ reagent kit manufactured by

Eppendorf (Westbury, NY) was employed in order to heat-denature the RNA. To each of

the two tubes were added 10 CIL liver or intestinal RNA (equivalent to 1 Gig) and 2 C1L

random decamers. The tubes were mixed, centrifuged briefly and heated for 3 min at 70-

850C in the thermocycler. Tubes were removed and left on ice for 1 minute. They were

centrifuged and put on ice again. The following components were added to each tube: 2

CIL 10xRT buffer, 1 C1L dNTP mix (10mM), 0.5 C1L RNase inhibitor, 1 C1L reverse

transcriptase, and RNase-free water to 20 C1L. The tubes were gently mixed and

centrifuged briefly. They were placed in the thermocycler for 1 hr at 42-440C, followed

by 920C for 10 min. The resulting cDNA was either stored at -200C or subjected to a

second round of PCR (liver, see below; for the intestine this procedure was performed a

few days after cDNA generation).

Degenerate primer design. A characteristic 'signature sequence', 44-amino acids

long, probably corresponding to the UDPGA binding site, has been shown to be highly

conserved amongst mammals and other vertebrates (Mackenzie et al., 1997). The relevant

amino acid and nucleotide sequences were compared in 4 species of fish using ClustalW.









The species investigated were Pleuronectes platessa UGTIBl, P.platessa UGT,

Pleuronectes yokohamnae UGT IB2, Epinephelus coiodes UGT and Danio rerio UGT.

Five primers were designed which could hypothetically bind to this sequence. The

application of exclusion criteria (degeneracy- <100-fold, poor or no matches with fish

sequences resulting from BLASTn searches, %GC content <40%, potential to self-

dimerize < -20 kcal/mol) resulted in the selection of two primers, designated as UGTR3

and UGTR4, and chosen to be reverse primers (Table 5-1). An additional reverse primer

(UGT RS) was chosen due to its low degeneracy (4-fold) and its complementarity to the

highly conserved N-terminal domain downstream of the signature sequence. Five

additional primers (UGTF3-7) were also chosen based on these same criteria. Since

these primers were complementary to sequences upstream of the signature sequence, they

were selected to be forward primers (Table 5-1).

Table 5-1. 5' 3' Sequences of degenerate primers chosen.

ID Sequence Direction

UGT F3 GTGGTSCTGGT SCCYGAAASYAGY Forward

UGT F4 CTTACWGAYCCMTTCYTKCC STGYGGC Forward

UGT F5 AAC AT GGTYYWWATYGGRGGYAT CAAC TGT Forward

UGT F6 ATYGGRGGYATCAACTGTGCA Forward

UGT F7 GAGT TTGT SVAHGGC TCW GGA Forward

UGT R3 AAAC AGHGGRAACAT CAVC AT Reverse

UGT R4 YC CYT GS TCK SCAAAC AGHGG Reverse

UGT R5 GT GRTAC TGRAT C CAGTT CAG Reverse









The primer pairs were selected in such a way that their melting temperatures did

not vary by more than 60C and their potential to heterodimerize was more than -20

kcal/mol (Table 5-2)

Table 5-2. Primer pairs chosen, showing annealing temperature and estimated amplicon
length


Pair Forward Reverse Amplicon length (bp)l T (oC)

3 UGT F6 UGT R5 618 52.6

4 UGT F7 UGT R3 288 53.8

5 UGT F6 UGT R3 339 53.8

6 UGT F7 UGT R5 567 52.6

7 UGT F5 UGT R4 363 61.1

8 UGT F3 UGT R4 1003 61.1

9 UGT F4 UGT R4 729 61.1



SBased on Danio rerio UGT sequence (Accession number NP_998587. 1)

PCR amplification of UGT cDNA. A 10 CIM solution of each primer in nuclease-

free water was made up. Each PCR tube consisted of 2 CIL DNA template (from catfish),

2 CIL forward primer, 2 C1L reverse primer, 0.5C1L Taq DNA polymerase (5U/C1L, in Mg

10x buffer), 1 CIL dNTP mix (10mM), 5 C1L 10xPCR buffer, and nuclease-free water up

to 50 CIL. Prior to the initiation of the PCR reaction, with the tubes in place, the

thermocycler lid was heated for two minutes at 1100C to prevent sample evaporation.

Thermocycler parameters (utilizing a gradient PCR program to adjust for the different

optimal annealing temperatures required by the various primer pairs) were as follows:










Stage Temp /oC Duration/min

Initial Denaturation 94 2

Denaturation 94 0.5

Annealing 5715 (L); 5515 (I)' 0.5

Extension 72 1.0

Final extension 72 7.0



annealing temperatures used for: L, liver; I, intestinal cDNA
The program consisted of 35 cycles of denaturation, annealing and extension.

The PCR products were subjected to electrophoresis on 1% agarose gel at 100V (in

lx TAE buffer (40 mM Tris base, 5 mM sodium acetate, 1 mM EDTA, pH 8.0)) using 30

CIL of PCR product; a 100bp DNA ladder was used for size estimates. The DNA bands

were visualized by placing on a UV transilluminator and recorded by photography.

Recovery of PCR product from gel and purification. The desired DNA band

was excised from the gel using a clean scalpel and transferred to a pre-weighed 1.5mL

microcentrifuge tube. The Wizard 9 SV Gel Clean Up system (Promega, Madison, WI)

was used to purify the PCR product by centrifugation. Membrane binding solution (4.5 M

guanidine isothiocyanate, 0.5 M potassium acetate, pH 5.0), 10 C1L per 10 mg gel, was

added to the gel slice. The mixture was vortexed and incubated at 510C for 10 min in

order to dissolve the gel slice. The tube was then briefly centrifuged at room temperature.

For every solution (derived from the cut gel slices), the following procedure was adopted.

One SV Minicolumn was placed in a collection tube. The dissolved gel mixture was

transferred to the SV Minicolumn assembly and incubated for 1 min at room temperature.

The assembly was centrifuged in a microcentrifuge at 16,000g for 1 minute and the liquid










in the collection tube was discarded. The column was washed by 700 C1L of Membrane

Wash Solution (10 mM potassium acetate, pH 5.0, 16.7 CIM EDTA, pH 8.0, 80%

ethanol). The assembly was centrifuged for 1 min at 16,000g, and the collection tube was

emptied. Another 500 CIL Membrane Wash Solution was added to the assembly, followed

by centrifugation for 5 minutes at 16,000g. The collection tube was emptied, and the

collection tube was recentrifuged for 1 minute to dry the column. The SV Minicolumn

was transferred to a clean 1.5 mL microcentrifuge tube and 50 CIL nuclease-free water

was applied to the column and incubated for 1 minute at room temperature. The

Minicolumn/micro-centrifuge tube was centrifuged for 1 minute at 16,000g. The

Minicolumn was discarded and the tube containing the eluted DNA was stored at -200C.

A portion of this DNA was diluted with 10 mM Tris-HC1, 1 mM EDTA, pH 8.0, and

used to calculate the DNA concentration by its absorbance at 260nm.

Ligation and transformation of E.coli. LB plates with ampicillin were first

prepared. LB Agar, 8.75 g, was weighed and dissolved in 250 mL, and the pH was

adjusted to 7.2 with NaOH. The solution was autoclaved for 30 min at 1200C. After the

medium cooled to around 500C, ampicillin was added to a final concentration of 100

Clg/mL. Some of the medium, 30-35 mL, was poured into 85-mm Petri dishes and the

agar left to harden. The plates were left overnight at room temperature and subsequently

stored in an inverted position at 40C

The ligation was performed using the p-GEM T-Easy Vector System@ supplied by

Promega. The volume of PCR product to be used in the ligation reaction could not exceed

3 CIL. The amount required was calculated from the following equation, which assumes

that the optimal insert:vector molar ratio is 3:1:










50 ng vector x a kb insert x 3 = ng insert required
3.0kb vector 1
where a is the approximate size of amplified insert

Because of this limit in sample volume, the amount of insert actually used was less

than that recommended since the concentration of purified DNA was relatively low. The

ligation reactions were setup as follows (all volumes in CIL) in 0.5 mL tubes:

Standard Positive Background
Component Reaction Control Control
2x Rapid Ligation Buffer, T4DNA ligase 5 5 5

pGEM T-Easy Vector (50 ng) 1 1 1

PCR-product (9 ng) 3----

Control insert DNA --- 2--

T4 DNA ligase (3 Weiss units/C1L) 1 1 1

DNase-free water 0 1 3

The ligation buffer was mixed vigorously before use. The reactions were mixed by

pipetting and incubated for 1 hour at room temperature, followed by storing overnight at

40C.

JM109 high-efficiency competent cells (>1x10s ofu/Clg DNA; Promega) were used

for transformation. The following procedure was performed using aseptic technique

(sterile tips and tubes, use of Bunsen flame to create upward convection in work area).

The tubes containing the ligation reactions were centrifuged for 1 minute at 10,000 rpm

and placed on ice. Another tube (transformation control, TC) was set up on ice; this

contained 0. 1 ng uncut plasmid (0. 1 CIL of 0. 1 mg/C1L solution used) in order to determine

the transformation efficiency of the competent cells. Tubes containing frozen aliquots of

JM109 cells were removed from -800C storage and placed on ice until thawed (~5 min).










The cells were mixed by gentle flicking of the tubes. Each ligation reaction, 2C1L, was

added to a 1.5 mL microcentrifuge tube on ice, followed by 50 CIL of cells (100 CIL were

added to the TC). The tubes were mixed by gentle flicking and left on ice for 20 minutes.

The cells were heat-shocked by placing in a 420C water bath for 45-50 sec. The tubes

were then returned to ice for 2 minutes. S.O.C medium (Invitrogen Corp., Carslbad, CA),

950 C1L, was added to each tube (900 CIL was added to the TC). The tubes were then

incubated for 1.5 h at 370C with shaking (~150 rpm). The ampicillin/LB plates were

removed from 40C storage, 100 CIL of 100 mM isopropylthiogalactoside (IPTG, a P-

galactosidase inducer) and 20 CIL of 50 mg/mL 5-bromo-4-chloro-3 -indolyl-P-D-

galactoside (X-Gal, hydrolyzed by P-galactosidase to yield a blue product) were added,

and the mixture spread on each plate. The agar was allowed to absorb these compounds

for 30 min at 370C. Samples, 100 CIL, of each transformation culture were transferred to,

and streaked on, duplicate LB/ampicillin/IPTG/ X-Gal plates; for the TC, 20C1L of tube

culture was diluted with 180 CIL of S.O.C. medium, and 100 C1L of this dilution was

applied to the agar plates. The plates were incubated overnight (~16h) at 370C. Plates

were then stored at 40C for 30 minutes to facilitate color development. The white

colonies should contain plasmids with the insert, while the blue colonies do not contain

the insert since the protein-encoding sequence of the lac Z gene in the vector is not

interrupted by the insert and hence can lead to P-galactosidase synthesis and catalysis of

the X-Gal reaction.

Colony PCR and culturing E.coli with insert of interest. Two white and one

blue colony from each plate were picked by a sterile wooden toothpick, which was

inserted in a PCR tube containing 5 CIL 10x PCR buffer, 5 CIL 10mM dNTP mix, 1 CIL









PUC/M13 forward primer, 1 CIL PUC/M13 reverse primer, 0.5 CIL Taq DNA polymerase,

and DNase-free water to 50 CIL. The pGEM T-Easy Vector contains binding sites for the

PUC/M13 primers. Thermocycler parameters were as shown previously, but with an

annealing temperature of 550C (no temperature gradient). PCR products were run on 1%

agarose gel at 100V, using 15 CIL of the PCR product.

Samples from colonies which showed the presence of insert on the gel were

extracted by an ethanol-flame sterilized metal hoop and dispensed into 14 mL sterile,

round-bottomed Falcon tubes containing 4 mL of LB medium with ampicillin by

swirling. The tubes were incubated with shaking for 16-20 h at 370C.

Purification of plasmid DNA. A sample, 850 CIL, of each culture medium was

diluted up to 1000 CIL with sterile glycerol and stored at -800C. The rest of the culture

medium was dispensed in 1.5 mL microcentrifuge tubes, which were centrifuged for 2

min at 10,000g. The supernatant was poured off and the tubes were blotted upside-down

on a paper towel to remove excess media. For plasmid purification, the Promega Wizard

Plus Minipreps 9 DNA Purification System was used. The cell pellets were resuspended

in 200 CIL of cell resuspension solution (50 mM Tris-HCI (pH 7.5), 10 mM EDTA, 100

Clg/mL RNase A). Cell lysis (0.2 M NaOH, 1% SDS) solution, 200 C1L, was added and

the tubes inverted 4 times to clarify the solution. Neutralization solution (1.32 M

potassium acetate, pH 4.8), 200 CIL, was added and mixed by inverting the tubes for 4

times, resulting in a white precipitate. The lysate was centrifuged at 10,000g for 5-20

minutes, depending on whether a cell pellet was clearly visible.

One Wizard@ Minicolumn was prepared for every Miniprep. A plunger was

removed from a 3 mL disposable syringe and set aside. The syringe barrel was attached









to the Luer-Lok@ extension of the Minicolumn. DNA purification resin (7 M guanidine

HC1), 1 mL, was pipetted in the barrel, followed by the cell lysate. The syringe plunger

was inserted in the barrel and used to push the slurry through the Minicolumn. The

syringe was detached from the Minicolumn and the plunger removed from the syringe

barrel. The barrel was then reattached to the Minicolumn. Column wash solution (80 mM

potassium acetate, 8.3 mM Tris-HCI (pH 7.5), 40 C1M EDTA, 55% ethanol), 2 mL, were

pipetted into the barrel of the Minicolumn/syringe assembly, and the solution was pushed

through the Minicolumn by the plunger. The syringe was removed, and the Minicolumn

was transferred to a 1.5 mL microcentrifuge tube, which was centrifuged at 10,000g for 2

min to dry the resin. The Minicolumn was transferred to a new 1.5 mL microcentrifuge

tube and 50 CIL nuclease-free water was added to the column and left for 1 minute. The

DNA was eluted by centrifuging at 10,000g for 20 sec. The Minicolumn was removed

and discarded, and the DNA solution stored at -200C. Products were visualized by 1%

agarose gel electrophoresis run at 100V, using 3 CIL of purified DNA and 10 CIL

quantitative 1kb plus DNA ladder (0.5 Gig).

Digestion with ecoRI. To ensure that the two DNA bands seen in the purified

plasmid DNA run on agarose gel were due to supercoiling of the DNA and not

contamination, the plasmid DNA was digested with the restriction enzyme ecoRI (pGEM

T-Easy Vector has restriction sites on either side of the insert). Plasmid DNA, 3 CIL, was

added to a tube containing 0.2 CIL acetylated BSA, 2 CIL 10x buffer, and 14.3 CIL DNase-

free water, and mixed by pipetting. ecoRI restriction enzyme (12 U/CIL), 0.5 CIL, was

added and the solution mixed by pipetting. The tubes were briefly centrifuged and









incubated for 2h at 370C. The products were run in 1% agarose at 100V, using all the

incubation mixture.

DNA sequencing and data processing. The concentration of the purified plasmid

DNA was determined prior to submission for sequencing. The DNA sequencing core

requires 1.5 Clg DNA for adequate processing. Cloned DNA sequences obtained were

then compared with nucleotide sequences in GenBank using the BLASTn tool provided

online (http://www.ncbi .nlm.nih.gov/BLAST). Multiple sequence comparisons were

done with SeqWeb, while two-sequence comparisons were done with the BLASTn 2.2.12

program.

Results and discussion (part 1)

Primer pairs 4 and 6 successfully amplified cDNA from catfish liver; while primer

pair 4 amplified cDNA from proximal intestine. The size of the amplicons were

approximately 300bp (pair 4) and 600bp (pair 6) in size, with the gel-clean up system

effectively removing primer dimers and other contamination (Figure 5-2). The controls

indicated that ligation and transformation of the plasmid into E.coli were successful.

Purified plasmid DNA was obtained from several colonies (Figure 5-3), which were

denoted as L1-L8 for the liver and 11-14 for the proximal intestine. The two bands seen in

these gels, did not represent contamination, as verified by the restriction digest of the

plasmid, which resulted in a band corresponding to the vector and one corresponding to

the smaller insert (Figure 5-4).










B


3 2 1 ladder ladder 3 2 1 ladder


500


Figure 5-2. Products of PCR reaction. 1(from intestine), 2 and 3 (from liver)

A. pre-cleanup; B. post-cleanup with the gel clean-up system. 100kb ladder
shown for size estimation.










Liver


L8 L7 L6 L5 L4 L3 L2 L1 1kb ladder


S2,500


Intestine


14 13 12 I11


1kb ladder


2,500


Figure 5-3. Plasmid DNA obtained from cultures transformed with vector containing
inserts from liver and intestine.









100bp ladder L8 L7 L6 L5 L4 L3 L2 L1 1kb ladder





~3,000


600
S300








Figure 5-4. Product of ecoRI digest of purified plasmids containing liver inserts L1-L8




The DNA sequences obtained are detailed in Appendix A. The results of the

BLASTn search (best five sequences for each insert) are summarized below (Table 5-3).

Very good matches were obtained with the Tetraodon nigroviridis cDNA, as well as

Pleuronectes yokohamnae UGT I B2, several Danio rerio sequences and

Strongylocentrotus purpuratus (sea-urchin) UGT2B sequences. There was also a good

similarity between the longer insert and the mammalian UGTIA sequences.

Better matches were obtained with the longer cDNA insert obtained from the liver

(95 sequences with score >50) than with the shorter insert from liver or intestine (9

sequences with score >50).











Table 5-3. Results of BLASTn search of cloned putative partial UGT sequences
Accession no. Short description Insert Score (bits) E-value
CNSOEYO6 Tetraodon nigroviridis, full length cDNA L1 123 3E-25
L4 115 8E-23
L7 113 6E-22
Il 107 2E-20

CNSOEVYF Tetraodon nigroviridis, full length cDNA L1 123 3E-25
L4 115 8E-23
L7 113 6E-22
Il 107 2E-20

AB120133.1 Pleuronectes yokohamae UGT1B2 mRNA L7 85.7 1E-13
L1 67.9 2E-08
L4 67.9 2E-08
Il 60.0 4E-06
AF104339 Macaca fascicularis UGT1A01, mRNA L7 63.9 5E-07
BC109404.1 Danio rerio cDNA clone L7 61.9 2E-06
BC100055.1 Danio rerio cDNA clone L1 60.0 4E-06
Il 52.0 1E-03
BX005348.9 Danio rerio DNA sequence from clone L1 60.0 4E-06
Il 52.0 1E-03
XM 792456.1 Strongylocentrotus purpuratus, UGT2B34 L4 54.0 3E-04
XM792428. 1 Strongylocentrotus purpuratus, UGT2B 17 L4 54.0 3E-04


SeqWeb analysis of the sequences showed that the short inserts were almost

identical with almost all the differences being located in the primer regions and thus may

be attributed to the degenerate nature of the primers. Sequence L1 was found to be 98%

similar to both sequences L7 and II. While this implied that all these sequences are

derived from the same isozyme, this could not be ascertained since most of the sequence

differences between UGT isoforms arise from the N-terminal (substrate-binding) domain

and only that part of the gene which codes for the highly conserved C-terminal domain

was cloned.









Methodology (part 2)

The next step in the cloning study was thus to design GSPs in order to extend the

partial UGT sequences obtained so far to the full-length gene.

Overview of RL~M-RACE

RNA-ligase mediated rapid amplification of cDNA ends, or RLM-RACE is a

procedure used to extend a known DNA sequence towards its 5'- and its 3'- ends

(Maruyama and Sugamo, 1994; Shaefer 1995).

In 5'-RACE, total RNA is treated with calf intestinal phosphatase (CIP) to remove

free 5'-phosphates from molecules such as ribosomal RNA, fragmented mRNA, tRNA,

and contaminating genomic DNA. The cap structure found on intact 5'-ends of mRNA is

not affected by CIP. The RNA is then treated with tobacco acid pyrophosphatase (TAP)

to remove the cap structure from full-length mRNA, leaving a 5'-monophosphate. A 45

base RNA Adapter oligonucleotide is ligated to the RNA population using T4 RNA

ligase. The adapter cannot ligate to dephosphorylated RNA because these molecules lack

the 5'-phosphate necessary for ligation. During the ligation reaction, the majority of the

full-length decapped mRNA acquires the adapter sequence as its 5'-end. A random-

primed reverse transcription reaction and nested PCR then amplifies the 5'-end of a

specific transcript (Figure 5-5). The Ambion kit used in this study provided two nested

primers corresponding to the 5'-RACE Adapter sequence, while two nested antisense

primers were designed to be specific to the target gene.

In 3'-RACE, first-strand cDNA is synthesized from total RNA using the supplied

3'-RACE Adapter. The cDNA is then subjected to PCR using one of the 3'-RACE

primers which are complimentary to the anchored adapter, and a user-supplied primer for

the gene under study (Figure 5-5). Although 3'-RACE may not require a nested PCR










reaction, this may also be performed if no significant amplicons are detected after the

outer PCR.


5' RLM-RACE

CIP treatment to remove 5'PO4
from degraded mRNA, rRNA,
tRNA, and DNA
CIP
5'-PO4~
G--P--P--P- AAAAA




TAP treatment to remove cap
from full-length mRNA


G--P--P--P- AAAAA




reverse transcription with
3' RACE Adapter


TAP
G--P-PI-P~ AAAA ~NVTTTTT-adapter
G--P- P AAAAG---P--P--P AAAAA


5' RACE Adapter Ligation to
decapped mRNA


PCR


5'-RACE adapter AAAAA




reverse transcription

5'-RACE adapter AAAAA

IIIIII


PCR

5'-RACE adapter


Figure 5-5. 5'- RLM-RACE and 3'- RACE


G--P--P--P NVTTTTT-adapter


3' RACE










Design of gene-specific primers (GSPs) for initial 5'-RACE study. The initial

primers used for RACE were designed to be 20-24 bases in length, with 50% G:C

content, and with no secondary structure. Primers contained less than 3G or C residues in

the 3'-most 5 bases, and did not have a terminal G at the 3'-end. An online

oligonucleotide analyzer (www.idtdna.com) was used to determine whether potential

primers self-hybridized or hybridized to the primers supplied with the RLM-RACE Kit.

Figure 5-6 shows where the gene-specific primers and the primers supplied with the kit

should be positioned with respect to the DNA template.

5' RACE

5' RACE 5' RACE UGT-specific
outer inner 5'primer
primer primer

5' RACE Adapter ~10bp



5'RACE UGT- 5'RACE UGT-
specific inner specific outer
primer primer

3' RACE

3'RACE UGT- 3'RACE UGT-
specific outer specific inner
primer primer

3' RACE Adiapter


3'RACE 3'RACE
inner outer
primer primer


Figure 5-6. Primer positions for 5'- and 3'-RACE.









The DNA templates selected were those identified from the previous study, that is,

the sequence isolated from liver (L6) and intestine (14). The primers used in this initial

study are shown in Table 5-4.

Table 5-4. Gene-specific primers used in initial 5'RLM-RACE study.

GSP ID Sequence (5' 3') Start positions PCR step

GSP OUT TGCTCTGAGGTCAGGTCGAA 397 Outer
GSP INN ACAGATACCCTCGTAGATGCCA 280 Inner
From 5' end of sense strand of partial sequence L6

Based on homology with the complete sequences of Pleuronectes platessa UGT1

(PPL249081) and M\'acaca fascicularis UGTIAl (AF104339) it was estimated that, for

the UGT sequence isolated from catfish liver, this sequence needed to be extend by ~920

bp to the 5' -end, and ~183 bp to the 3'-end. Unfortunately, the use of these primers led

to sequences which still lacked the 5'-end (L15R, L25R, L35R, Il5R, I25R, I35R; see

Appendix A). In addition, a high degree of non-specific binding was noted.

Design of GSPs for succeeding 5'- and 3'-RACE study. A new batch of GSPs

was designed (Table 5-5) using different criteria than the ones mentioned above in an

attempt to improve sensitivity. Primer 3.0 Software (http://frodo.wi .mit.edu/cgibin/

primer3/primer3_wYww.cgi) was used to design primers, based on the following criteria:

a. For 5'-RACE, GSPs with a GC-clamp at the 3'-end in order to reduce non-

specific binding were used,

b. The outer and inner primer melting temperatures for the GSPs were within a

degree of the RACE kit supplied primers,

c. For 5'-RACE, the inner primer was long (~27 bp) in order to reduce nonspecific

binding, and











d. The primers were designed to anneal close (50-75 bp) to the existing 5'-end to

avoid large overlaps.

Different sets of primers were designed based on the cDNAs obtained by the study

involving the degenerate primers (I4) and the initial 5'-RACE study (I3 5R and L25R).




Table 5-5. Gene-specific primers used in succeeding RLM-RACE study


GSP ID


Sequence (5'-3')


RACE Start' PCR step


(a) Liver L25R

UGT 50UT1
UGT 50041

UGT 30UT1
UGT 3IbW1

(b) Intestine I4
UGT 50UT2
UGT 50UT2A
UGT 5INN2
UGT 5HOWLL

UGT 30UT2
UGT 30UT2A
UGT 3HItz
UGT 3HOWLL


ATTGGGCATTACAGGTCTCG
CGAGGACGTCTCTGAACGTAACATCC


119 Outer
51 Innel

320 Outer
351 Inner


GATTCCTCAGAGGGTTCTGT
GGGGTCATTCCCAAAGACAT


GCCGTTACAGATACCCTCGT
GTATCGCCACAGAACCCTCT
ACACGAAGGAGCTCAAAGTGAACACG
GCCACTTCATCACTTTGACATTTTCAGG


Outer
Outer
Inmer
Imler

Outer
Outer
Imier
Imier


AATGTCAAAGTGATGAAGTGG
GACATTCCTGAAAATGTCAAA
CCCAAGGCTAAGGTGTTCATC
GACCTCTTAGCACACCCCAAG


(b) Intestine I35R


UGT 50UT3
UGT 50UT3A
UGT 5RIN3
UGT 5HIthk

UGT 30UT3
UGT 30UT3A
UGT 3DIN3
UGT 3INN2A


TGTTAATGACCTTCGGTGTGA
ATGACCTTCGGTGTGAGTTTT
AAACCTAAGAGGTCATTCTGCGGAAGC
ATGGGGACCGGGTGTCTATTTATTACG

TTTCCAGCTAACACTACTTGG
TTACACGTCCTCTAACCGTAA
CCCAAGGCTAAGGTGTTCATC
CCATGGCATCTACGAGGGTAT


Outer
Outer
Innel
Imier

Outer
Outer
Innel
Inner


SStart position from partial DNA sequences obtained so far









5' RLM-RACE procedure

Calf intestinal phosphatase (CIP) treatment. Total RNA (not DNase treated) (2

CIL for liver; 1 CIL for intestine), 10 Gig, as well as 10 Clg of control RNA (mouse thymus)

were gently mixed with CIP buffer, CIP, and nuclease-free water in a total volume of 10

CIL. The mixture was incubated at 370C for 1 hour, and terminated by the addition of 15

CIL ammonium acetate solution. A 115 CIL volume of nuclease-free water was added,

followed by 150 CIL acid phenol-chloroform. The mixture was then vortexed thoroughly

and centrifuged for 5 minutes at room temperature and at > 10,000g. The aqueous phase

was transferred to a new tube, 150 CIL chloroform were added, and the mixture was

thoroughly vortexed and centrifuged for 5 minutes at > 10,000g. The top aqueous layer

was transferred to a new tube, 150 C1L isopropanol were added, followed by thorough

vortexing and chilling on ice for 10 minutes. The mixture was then centrifuged at

maximum speed (16,000g) for 20 minutes. The pellet was rinsed with 0.5 mL cold 70%

ethanol and centrifuged for 5 minutes at 16,000g. The ethanol was carefully removed and

discarded, and the pellet was allowed to air dry (but not completely). The pellet was

resuspended in 11 CIL nuclease-free water and placed on ice. At this point 1 CIL of the

CIP-treated RNA was reserved for the "minus-TAP" control reaction. This RNA was

carried through adapter ligation, reverse transcription and PCR in order to demonstrate

that the products generated by RLM-RACE were specific to the 5'-ends of decapped

RNA.

Tobacco Acid Pyrophosphatase (TAP) treatment. CIP'd RNA, 5 C1L, was gently

mixed with TAP, 10XTAP buffer and nuclease-free water in a total volume of 10 CL.

The mixture was incubated at 370C for 1 hour.









5'RACE Adapter Ligation. CIP/TAP-treated RNA, 2 CIL, and 2 CIL of CIP-treated

RNA (minus-TAP control) was gently mixed with 1 CL 5'RACE adapter (5'-

GCUGAUGGCGAUGAAUGAACACUGCGU7UUGCUGGCU7UUGUAA-3) 1

CIL 10XRNA Ligase buffer (before use, the buffer was quickly warmed by rolling it

between gloved hands to resuspend any precipitate), T4 DNA Ligase (2.5 U/CIL), and

nuclease-free water in a total volume of 10 C1L. The mixture was incubated at 370C for 1

hour, after which it was stored at -200C.

Reverse transcription (RT). Ligated RNA, 2C1L, or minus-TAP control were

gently mixed with 4 CIL dNTP mix, 2 CIL random decamers, 2 CIL 10XRT buffer, 1 C1L

RNase inhibitor, 1 CIL M-MLV reverse transcriptase, and nuclease-free water in a total

volume of 20 CIL. The mixture was incubated at 420C (or 500C, see results) for 1 hour.

The reactions were stored at -200C.

Outer PCR. Each tube contained: 1 CIL RT reaction, 5 C1L 10XPCR buffer, 4 C1L

dNTPmix (4 mM), 2 CIL gene-specific or outer control (reverse) primer (10 CIM), 2 C1L

outer (forward) p ri mer ( 10 CM) (5'- GC TGAT GGC GAT GAAT GAAC AC TG-3'), 0.2 5

CIL Taq DNA polymerase (5 U/C1L), and nuclease-free water in a total volume of 50 CL.

A minus-template control was also included to ensure that one or more of the PCR

reagents was not contaminated with DNA.









Thermocycler parameters were as follows (Lid heating at 1100C):

Step Stage Temp/oC Duration/min

1 Initial denaturation 94 3
2 Denaturation 94 0.5
3 Annealing 59 + 21 0.5
4 Extension 722 13
5 Final extension 72 7

35 cycles of steps 2 4 were performed
1,2,3 These parameters were frequently changed to optimize the PCR. The values given
above are representative of parameters used with the GSP_OUT and control primers.

Inner nested PCR. A mixture was prepared, identical to the one for outer PCR,

except that the DNA template was now 1 C1L of the outer PCR, and 2 C1L each of both

inner primers. The sequence for the inner 5'RACE primer supplied with the kit was 5'-

CGC GGATCCGAACACTGCGTTTGC TGGCTTTGATG -3'. The thermocycler

parameters were similar except for the annealing temperature, which was typically higher

than the one used for the outer PCR.

3' RACE procedure

Reverse transcription. The following components were assembled in a nuclease-

free microfuge tube: 1 Clg total RNA (DNase-treated) from intestine or liver or control

(mouse thymus RNA), 4 CIL dNTP mix, 2 CIL 3'RACE Adapter (5'

GCGAGCACAGAATTAATACGACTCACTATAGG T12VN 3'), 2 CIL 10XRT buffer,

1 CIL RNase inhibitor, 1 C1L M-MLV reverse transcriptase, and nuclease-free water to 20

CIL. The reaction was mixed gently and incubated at 420C or 500C for 1 hour.

PCR. The procedure for the outer and inner PCR was similar to the one performed

for 5'-RACE, the only difference being the GSP and the kit-supplied primers used. The










sequences for the letters were as follows: Outer 5' -GC GAGCACAGAATTAATACGA

CT-3', Inner 5' -CGCGGATCCGAATTAATACGACTCACTATAGG-3'`

PCR amplification of entire UGT gene

Elucidation of the complete gene sequence for liver UGT from catfish by RLM-

RACE (via partial sequence overlap) enabled the design of gene specific primers which

are complementary to the gene itself as well as the untranslated region. The primers used

are shown in Table 5-6. All primers complementary to the untranslated region (UTR)

were designed with the help of Primer3 software, except for the pair of primers that were

complementary to the exact start and end of the gene (LIVUGTF1 and LIVUGTR1

respectively).


Table 5-6. Primers used for amplifying liver and intestinal UGT gene

GSP ID Sequence (5' 3') Start position

UTR Fl CTGCTTCCTCTAGACGTAATTAGAAAC 40
UTR F2 CTCACATTCCTCCTCCTTCTTTTT 76
UTR R1 GAAC GT GGT GAT GAGAACACTATAACT + 121
UTR R2 TAGTGACATCATAACAACCGTAACTGC + 190
LIVUGT Fl ATGCCTCGTCTTCTTGCAGCTCTCTGT 1
LIVUGT R1 TCACTCCTTTTTGCTCTTCTGAGCCCT 1568

Due to the length of the amplicon (~1.6kb), Super Taq Plus polymerase (Ambion

Inc) was employed. This enzyme results in higher yields with amplicons >1kb. In

addition, this enzyme mixture has a proof-reading ability, which will be important for

future expression of the gene, as well as providing greater fidelity and processivity than

ordinary thermos Taq DNA polymerase. An extension temperature of 680C and an

extension time of 1.75 min were used for this PCR. Different combinations of UTR




Full Text

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PHASE II BIOTRANSFORMATION OF XENOBIOTICS IN POLAR BEAR ( Ursus maritimus ) AND CHANNEL CATFISH ( Ictalurus punctatus ) By JAMES C. SACCO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by JAMES C. SACCO

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This document is dedicated to Denise and my parents.

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iv ACKNOWLEDGMENTS First and foremost, I would like to thank my mentor, Dr. Margaret O. James, for her instruction, guidance, and support throughout my PhD program. Through her excellent scientific and mentoring skills I not only managed to complete several interesting studies but also rekindled my scientific curiosity with regards to biotransformation and biochemistry in gene ral. I greatly appreciate the advice and instruction of Ms. Laura Fa ux, our laboratory manager, on enzyme assays, HPLC, and fish dissection. The assistance and advice of Dr. David S. Barber, Mr. Alex McNally, and Mr. Jason Blum at the Center for Huma n and Environmental Toxicology in walking me through the complexities of molecula r cloning are much appreciated. Academic discussions with Dr. Liquan Wang, Dr. Ke n Sloan, Dr. Joe Griffitt and Dr. Nancy Denslow also helped me to interpret my results and design better experiments accordingly. Last but not least, I would like to tha nk my fiancée, Denise, and my parents, for their support and encouragement throughout my doctoral studies.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix ABSTRACT......................................................................................................................x ii CHAPTER 1 BIOTRANSFORMATION AND ITS IMPORTANCE IN THE DETOXIFICATION OF XENOBIOTICS...................................................................1 2 PHASE II CONJUGATION: GLUCUR ONIDATION AND SULFONATION.........4 UDP-Glucuronosyltransferases (UGTs).......................................................................7 Sulfotransferases (SULTs)..........................................................................................11 3 SULFONATION OF XENOBIOTICS BY POLAR BEAR LIVER.........................14 Hypothesis..................................................................................................................17 Methodology...............................................................................................................17 Results........................................................................................................................ .23 Discussion...................................................................................................................32 Conclusions.................................................................................................................37 4 GLUCURONIDATION OF POLYCHLORINATED BIPHENYLOLS BY CHANNEL CATFISH LIVER AND INTESTINE....................................................38 Hypothesis..................................................................................................................41 Methodology...............................................................................................................41 Results........................................................................................................................ .44 Discussion...................................................................................................................53 Conclusions and Recommendations...........................................................................59 5 CLONING OF UDP-GLUCURONOSYLTRANSFERASES FROM CHANNEL CATFISH LIVER AND INTESTINE........................................................................60 Piscine UGT Gene Structure and Isoforms................................................................60

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vi Hypothesis..................................................................................................................62 Methodology (part 1)..................................................................................................62 Results and discussion (part 1)...................................................................................74 Methodology (part 2)..................................................................................................79 Overview of RLM-RACE...................................................................................79 5` RLM-RACE procedure...................................................................................84 3` RACE procedure.............................................................................................86 PCR amplification of entire UGT gene...............................................................87 Results (part 2)............................................................................................................89 Nucleotide sequence analysis..............................................................................89 Protein sequence analysis..................................................................................100 Cloning of entire UGT gene..............................................................................104 Discussion.................................................................................................................107 Limitations................................................................................................................110 Conclusions and recommendations..........................................................................114 6 DETERMINATION OF PHYSIOLOGICA L UDPGA CONCENTRATIONS IN CHANNEL CATFISH LIVER AND INTESTINE..................................................116 UDP-Glucuronic Acid (UDPGA).............................................................................116 Objective...................................................................................................................118 Method Development...............................................................................................118 Sample Digestion...............................................................................................119 HPLC.................................................................................................................121 Final Method.....................................................................................................123 Results.......................................................................................................................1 25 Discussion.................................................................................................................127 Conclusions and Recommendations.........................................................................129 APPENDIX A SEQUENCES OF UGT PARTIAL CLONES AND AMPLICONS........................131 B SEQUENCES FOR UGT FU LL-LENGTH CLONES FROM CATFISH LIVER..138 LIST OF REFERENCES.................................................................................................144 BIOGRAPHICAL SKETCH...........................................................................................157

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vii LIST OF TABLES Table page 2-1 Expression of human UG T mRNA in various tissues................................................9 2-2 Tissue distribution of SULTs (cDNA and mRNA) in humans................................12 3-1 Estimated kinetic parameters (Mean SD) for (a) sulfonation and (b) glucuronidation of 3-OH-B[a]P by polar bear liver cytosol and microsomes.........24 3-2 Kinetic parameters (Mean SD) for the sulfonation of various xenobiotics by polar bear liver cytosol, listed in orde r of decreasing enzymatic efficiency............27 4-1 Estimated kinetic parameters (mean ± S.D.) for the co-substrate UDPGA in the glucuronidation of thr ee different OH-PCBs...........................................................44 4-2 Kinetic parameters (Mean ± S.D.) fo r the glucuronidation of 4-OHBP and OHPCBs.........................................................................................................................48 4-3 Comparison of the estimated kinetic pa rameters for OH-PCB glucuronidation in catfish liver and proximal intestine..........................................................................48 4-4 Comparison of kinetic parameters (M ean ± SEM) for the glucuronidation of OHPCBs grouped according to the numb er of chlorine atoms flanking the phenolic group..........................................................................................................49 4-5 Results of regression anal ysis performed in order to investigate the relationship between the glucuronidation of OH-PCBs by catfish proximal intestine and liver and various estimated physical parameters..............................................................52 5-1 5' 3' Sequences of degenerate primers chosen.......................................................66 5-2 Primer pairs chosen, showing annealing temperature and estimated amplicon length........................................................................................................................6 7 5-3 Results of BLASTn search of cloned putative partial UGT sequences...................78 5-4 Gene-specific primers used in initial 5`RLM-RACE study.....................................82 5-5 Gene-specific primers used in succeeding RLM-RACE study................................83 5-6 Primers used for amplifying liver and intestinal UGT gene....................................87

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viii 5-7 Results of blastn search for livUGTn (and intUGTn)..............................................92 5-8 Promoter prediction..................................................................................................92 5-9 Results of blastp search for liv/intUGTp..................................................................94 5-10 Results of blastn search for I35R_C.........................................................................99 5-11 Potential antigeni c sites on liv/intUGTp................................................................101 5-12 Results of blastp search for I35R_Cp.....................................................................102 5-13 Results of ClustalW multiple sequence alignment analysis of the cloned UGTs and the original livUGTn.......................................................................................106 5-14 Conserved consecutive residues obser ved in catfish liver and mammalian UGTs (sequences shown in Figure 5-13)..........................................................................109 6-1 UDPGA concentrations ( M) in liver and intest ine of various species.................117 6-2 Elution times of certain physiological s ubstances (standards dissolved in mobile phase) using the anion-exchange HPLC conditions described above....................124 6-3 UDPGA concentrations in M (duplicates for individual fish), in catfish liver and intestine............................................................................................................126

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ix LIST OF FIGURES Figure page 1-1 Schematic of select xenobiotic (represented by hydroxynaphthalene) biotransformation pathways in the mammalian cell..................................................3 2-1 Structure of the co-substrates PAPS and UDPGA (transferred moieties shown in bold) and the formation of the polar sulfonate and glucur onide conjugates, shown here competing for the same substrate............................................................6 2-2 Proposed structure of UGT, based on amino acid sequence......................................7 2-3 Complete human UGT1 complex locus re presented as an array of 13 linearly arranged first exons..................................................................................................10 2-4 The human UGT2 family.........................................................................................10 3-1 Structures of sulfonation substr ates investigated in this study.................................15 3-2 Sulfonation of 3-OH-B[ a]P at PAPS = 0.02 mM.....................................................25 3-3 Eadie-Hofstee plot for the glucur onidation of 10 M 3-OH-B[a]P, over a UDPGA concentration ra nge of 5-3000 M............................................................26 3-4 Sulfonation of 4-OH-PCB79, PAPS = 0.02 mM....................................................28 3-5 Autoradiogram showing the revers e-phase TLC separation of sulfonation products of OHMXC................................................................................................29 3-6 Autoradiogram showing the revers e-phase TLC separation of sulfonation products from incubations with TCPM....................................................................30 3-7 Autoradiogram showing the revers e-phase TLC separation of sulfonation products of TCPM and the eff ect of sulfatase treatment..........................................31 3-8 Autoradiogram showing reverse-phase TLC separation of sulfonation products from the study of PCP kinetics.................................................................................32 4-1 Structure of substrates used in channel catfish glucuronidation study.....................42

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x 4-2 UDPGA glucuronidation ki netics in 4 catfish..........................................................46 4-3 Representative kinetics of the gluc uronidation of OH-PCBs in 4 catfish................47 4-4 Decrease in Vmax with addition of second chlorine atom flanking the phenolic group, while keeping the chlorine su bstitution pattern on the nonphenolic ring constant.....................................................................................................................50 4-5 Relationship between Vmax for OH-PCB glucuronidation in intestine and liver and ovality...............................................................................................................53 5-1 Summary of methods used to clone channel catfish UGT.......................................63 5-2 Products of PCR reaction. 1(from intestine), 2 and 3 (from liver)...........................75 5-3 Plasmid DNA obtained from cultures tran sformed with vector containing inserts from liver and intestine............................................................................................76 5-4 Product of ecoRI digest of purified pl asmids containing liver inserts L1-L8..........77 5-5 5 RLM-RACE and 3 RACE................................................................................80 5-6 Primer positions for 5 and 3 -RACE......................................................................81 5-7 Full nucleotide sequence obtained for hepatic catfish UGT (livUGTn), derived from 4 sequencing runs each....................................................................................90 5-8 Sizes and positions of partial UGT sequences (cross-hatched rectangles) from intestine and liver, corresponding to two di stinct isoforms, relative to complete sequences for liver and intestin al UGT (solid rectangles).......................................91 5-9 Identification of open read ing frame using ORF Finder..........................................93 5-10 Predicted protein sequence liv/intUGTp from liv/intUGTn....................................93 5-11 Comparison of liv/intUGTp with homol ogous proteins in other fish, showing scores and alignment of closely related sequences.................................................95 5-12 Phylogram for fish UGT prot eins homologous to liv/intUGTp...............................96 5-13 Alignment of liv/intUGTp (excluding UTRs) with selected mammalian UGT proteins, showing scores and multiple alignment of sequences, highlighting important regions and residues (see discussion)......................................................97 5-14 Phylogram for I.punctatus liv/intUGTp and selected mammalian UGT proteins...98 5-15 Multiple sequence alignment between livUGTn and I35R_C.................................99 5-16 Results of NCBI conserved domain search............................................................100

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xi 5-17 Kyte-Doolittle Hydrophobici ty Plot for liv/intUGTp............................................101 5-18 Results of NCBI conserved domain search for I35R_Cp......................................103 5-19 Alignment of predicted protein sequen ces from cloned catfish UGTs. Regions of interest and the starting and ending residue of the mature product are highlighted..............................................................................................................104 5-20 Cloning of livUGTn...............................................................................................105 5-21 Cloning of intUGTn...............................................................................................105 5-22 Multiple sequence alignment for fish sequences homologous to catfish UGT isolated from liver and intestine, show ing regions where substrate binding of phenols is thought to occur for mammalian UGT1A isozymes.............................110 5-23 Results of 3 RACE performed on liver, showing multiple products obtained......111 5-24 3 RACE for I4.......................................................................................................112 5-25 PCR amplification of UGT using degenerate primers...........................................114 6-1 Heat-induced degradation of UDPGA (boiling in 0.25 M H2PO4 buffer).............120 6-2 Decomposition of UDPGA to UDP and UMP after boiling in 0.25 M H2PO4 buffer for 10 minutes..............................................................................................120 6-3 Effect of boiling liver tissue for 1 mi nute in two different concentrations of buffer. A, 0.25 M H2PO4, p H 3.4; B, 0.30 M H2PO4, p H 4.3................................121 6-4 HPLC chromatogram for catfish AT17 liv er. Center refers to region of liver from which the sample was taken..........................................................................122 6-5 HPLC chromatogram for catfish AT18 in testine. Rep 2 refers to second sample taken from AT18 intestine......................................................................................123 6-6 HPLC chromatogram of UDP, UDPgalacturonic acid (UDPGTA), and UDPGA standards.................................................................................................................125 6-7 Comparison of hepatic and intestinal [U DPGA] in 4 individual channel catfish..126

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xii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHASE II BIOTRANSFORMATION OF XENOBIOTICS BY POLAR BEAR ( Ursus maritimus ) AND CHANNEL CATFISH ( Ictalurus punctatus ) By James C. Sacco August 2006 Chair: Margaret O. James Major Department: Medicinal Chemistry Both polar bears and channel catfish are s ubject to bioaccumula tion of persistent toxic environmental pollutants including hydroxylated compounds, which are potential substrates for detoxification via phase II c onjugative processes such as sulfonation and glucuronidation. The objectives of this diss ertation were to (a) study the capability of polar bear liver to sulfonate a structurally diverse group of environmental chemicals, and to study the glucuronidation of 3-OH-B[a]P; (b) study the effect s of chlorine substitution pattern on the glucuronidation of polychlorin ated biphenylols (OH-PC Bs) by catfish liver and proximal intestine; (c) clone UDP-glucur onosyltransferase (UGT) from catfish liver and intestine; (d) de velop a method to determine physio logical concentrations of UDPglucuronic acid (UDPGA) in cat fish liver and intestine. In the polar bear, the efficiency of sulfonation decreased in the order 3-OHB[a]P>>>triclosan>>4´-OH-PCB79>OHM XC>4´-OH-PCB165>TCPM>4´-OH-PCB159 >PCP, all of which produced detectable su lfate conjugates. Substrate inhibition was

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xiii observed for the sulfonation of 3-OH-B[a] P and 4´-OH-PCB79. The hexachlorinated OH-PCBs, TCPM and PCP were poor substrates for sulfonation, suggesting that this may be one reason why these substances and struct urally similar xenobiotics persist in polar bears. OH-PCBs are glucuronidated with similar efficiency by channel catfish liver and proximal intestine. There were differences in the UGT activity profile in both organs. Both hepatic glucuronidation and intestinal glucuronidation were decreased with the addition of a second chlorine atom flanking the phenolic group, whic h is an arrangement typical of toxic OH-PCBs that persist in organisms. One full length UGT from catfish liver, together with a full-length UGT (identical to the liver UGT), and a partial sequence of a different UGT from catfish intestine were cloned. The full-length catfish UGT clone appeared to be analogous to mammalian UGT1A1 or UGT1A6. The anion-exchange HPLC method devel oped to determine UDPGA was sensitive, reproducible and displayed good resolution fo r the co-substrate. The hepatic UDPGA levels determined by this method were simila r to those in other mammalian species and higher than reported for two ot her fish species. This was th e first time intestinal UDPGA concentrations in any piscine species were determined; the values were similar to rat intestine, but significantly higher than in human small intestine.

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1 CHAPTER 1 BIOTRANSFORMATION AND ITS IMPORT ANCE IN THE DETOXIFICATION OF XENOBIOTICS The exposure of biological systems to environmental compounds which may be potentially toxic to these systems has spurre d the evolution of an elaborate, protective biochemical system whereby these xenobiotic s are eliminated from cells and whole organisms, usually via chemical transformatio n (or biotransformation). This system is composed of a multitude of enzymes, which while being distributed in many tissues and organs, are principally located in organs such as liver, inte stine and lungs. This is of physiological significance since these tissues represent major routes of xenobiotic entry into organisms. Within cells, biotransfo rmation enzymes also display a level of organization in that while some are soluble and found in the cytosol (e.g. sulfotransferases (SULT), glutat hione-S-transferases), others are relatively immobile and membrane-bound (e.g. UDP-glucuronosyltransf erases (UGT) and cytochrome P450s (CYP) in the endoplasmic reticulum). Since it is highly improbable that the orga nism has a substrate-specific enzyme for metabolizing every potential xenobiotic, biotransformatio n enzymes are generally nonspecific, acting on a broad range of structurally unrelated su bstrates. In addition, several isoforms of the same enzyme (or more th an one enzyme) may catalyze product formation from the same substrate, albeit at different ra tes and with different affinities. Enzymes in the same superfamily as those that ac t upon xenobiotics can also biotransform endogenous substances, indicating an equall y important regulatory role for these

PAGE 15

2 enzymes. This interrelationship between different enzymes and substrates can be illustrated by the metabolism of -estradiol in humans, which can be biotransformed both via sulfonation (SULT1E1, which also acts on 7-hydroxymethyl -12-dimethylbenzanthracene, the product of CYP450-catalyzed hydroxylati on of 7,12-dimethyldibenzanthracene (Glatt et al., 1995)) and glucuronid ation (UGT1A1, which can also conjugate 1-naphthol (Radominska-Pandya et al., 1999)). While these enzymes mainly represent a cellular defense mechanism against toxicity, occasionally procarci nogenic and protoxic xenobiotics are metabolized to active metabolites that attack macromolecules su ch as DNA, proteins and lipids. In exposed organisms, metabolism is an important factor in determining the bioaccumulation, fate, toxicokinetics, and toxic ity of contaminants. The majority of the compounds of interest to this study ar e derived from Phase I metabolism of environmental pollutants. These metabolites ha ve been shown to have toxic effects both in vitro and in vivo , effects that can be eliminated by Phase II biotransformation (Chapter 2). In addition, contaminant exposure can resu lt in the induction or inhibition of both Phase I and Phase II enzymes. For exampl e, induction of CYP 1A (e.g., by polyaromatic hydrocarbons (PAHs) or co-planar polychlor inated biphenyls (PCBs)), CYP 2B and CYP3A (e.g., by o -chlorine substituted PCBs) will lead to increased formation of hydroxylated metabolites. Thus, a balance be tween the CYP and conjugative Phase II enzymes, sometimes directly mediated by the xenobiotic substrates and/or their metabolites, is responsible for either the detoxification or the accumulation of toxic metabolites in the body. The final removal of these metabolites from the cell is brought about by several different gr oups of membrane proteins (e .g., organic anion transport

PAGE 16

3 protein (OATP), multidrug-resistance associated protein (MRP)), a process sometimes referred to as Phase III biot ransformation (Figure 1-1). OH O S O O O O OH OH OH O O O OH OH OH O OH S O O O O OH OH OH O O O OH OH OH OH CYP UGT SULT UGT cytosol ER lumen ER membrane MRP OATP MRP OATP Figure 1-1. Schematic of select xenobi otic (represented by hydroxynaphthalene) biotransformation pathways in the mamma lian cell. For abbreviations see text.

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4 CHAPTER 2 PHASE II CONJUGATION: GLUCURONIDATION AND SULFONATION Biotransformation has been conveniently categorized into two distinct phases. While the consecutive numbering of these pr ocesses implies a sequence, this is not always the case and the extent of involvement of both phas es in the metabolism of a compound depends on both its chemical stru cture and physical properties. Phase I biotransformation usually consists of oxidatio ns carried out largely by CYP enzymes and flavin monooxygenases and hydrolysis reactio ns executed by ester hydrolase, amidase and epoxide hydrolase (EH). A variety of chemi cal moieties can be conj ugated to suitable acceptor groups on xenobiotics as part of Phase II biotransformation, including glucuronic acid (UGT), sulfonic acid (SU LT), glutathione (GST), amino acids, and an acetyl group (N-acetyltransferase). With the exception of acetylation, methylation and fatty acid conjugation, the strategy of Phase II biotransformation is to convert a xenobiotic to a more hydrophilic form via the attachment of a chemical moiety which is ionizable at physiological p H. The resulting anionic conjugate is then readily excreted in bile, feces, or urine, and is generally unable to undergo passive penetr ation of cell membranes. This metabolic transformation also results in reduced affi nity of the compound fo r its cellular target. Enterohepatic recycling may result in the hydr olysis of biliary ex creted conjugates and the regeneration of the pa rent compound, which is then subject again to biotransformation after being reabsorbed through the gut mucosa. In a few cases, the

PAGE 18

5 conjugate is pharmacologically active, as in the case of mor phine-6-glucuronide (Yoshimura et al., 1973) and minoxi dil sulfate (Buhl et al., 1990). The moieties attached to the xenobi otic in the case of sulfonation and glucuronidation are a sulfonate group ( p Ka 2) or glucuronic acid ( p Ka 4-5). The cosubstrates which supply these highly polar species are, respectiv ely, 3´-phosphoadenosyl5´-phosphosulfate (PAPS) and uridine 5´-d iphosphoglucuronic acid (UDPGA) (Figure 21). The mechanism of both reactions, whic h occurs as a ternary complex, is a SN2 reaction, the deprotonated acceptor group of the substrate attacking the sulfur in the phosphosulfate bond of PAPS, or the C1 of the pyranose ring to which UDP is attached in an -glycosidic bond in the case of UDPGA. The resulting c onjugates are then released. PAP and UDP also leave the enzyme’s active site and are subsequently regenerated. There may be competition for the same acceptor group, especially for phenols. Other acceptor groups that can be conj ugated by both processe s include alcohols, aromatic amines and thiols. Glucuronidati on is also active on other functional groups, including carboxylic acids, hydroxylamines, a liphatic amines, sulfonamides and the C2 of 1,3-dicarbonyl compounds. SULTs are ge nerally high-affinity, low-capacity biotransformation enzymes that operate effec tively at low substrate concentrations. Thus, typical Kms for the sulfonation of xenobiotic subs trates are usually significantly lower than Kms for the same substrates undergoing biot ransformation by the low-affinity, highcapacity UGTs. For example, kinetic parame ters for the sulfonation and glucuronidation of the antimicrobial agent triclosan in human liver are Km values of 8.5 and 107 M and Vmax of 96 and 739 pmol/min/mg protein re spectively (Wang et al., 2004).

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6 N OH O H O O O OH OH O H O O H P O O OH P O OH N O O H O N N N NH2 N OH O O O P O O H O S O H O O P O H O O H OH Cl O Cl S O O O OH OH OH O O O O Cl UDPGA PAPS PAPS PAP SULT (cytosol) UDPGA UDP UGT (ER) sulfonate conjugate glucuronide conjugate xenobiotic Figure 2-1. Structure of the co-substrates PAPS and UDPGA (transferred moieties shown in bold) and the formation of the polar sulfonate and gluc uronide conjugates, shown here competing for the same substrate.

PAGE 20

7 UDP-Glucuronosyltransferases (UGTs) The primary sequence of human UGTs ra nges from 529 to 534 amino acids in length (Tukey and Strassburg 2000). These 50-56 kDa proteins reside in the endoplasmic reticulum, whereby the amino terminus and around 95% of the subsequent residues are located in the lumen. A 17-amino acid-long transmembrane segment connects the lumenal part of the enzyme with the short (19-24 residues) carboxyl-terminus located in the cytosol (Figure 2-2). The active enzyme pr obably consists of dimers, linked together at the C-terminus (Meech and Mackenzie 1997). The existence of tetramers for the formation of the diglucuronide of B[a]P-3,6-diphenol has be en suggested (Gschaidmeier and Bock 1994). COO-NH3 +Aglycone+ +Cytosol ER membrane ER lumenUDPGA Figure 2-2. Proposed structure of UGT, based on amino acid sequence Based on evolutionary divergence, mammalian UGTs have been classified into four distinct families (Mackenzie et al., 2005): fa mily 1, which includes bilirubin, thyroxine and phenol UGTs; family 2, which includes steroid UGTs; family 3, which includes UGTs whose substrate specificity is, as yet, unknown (Mackenzie et al., 1997); family 8, represented by UGT8A1 which utilizes UDP-galac tose as the sugar donor (Ichikawa et

PAGE 21

8 al., 1996). Although the liver is the major site of glucuronidation in the living organism, several other tissues have been shown to expr ess UGTs. The small intestine appears to be an equally important site of glucuronidation, particularly for ingested xenobiotics. In addition, expression of some UGT isofor ms is tissue-specific (Table 2-1). The nine family 1 UGT isoforms (UGT1) are all encoded by one gene that has multiple unique exons located upstream of four common exons on human chromosome 2q37 (Figure 2-3). The isoforms are generated by differential splici ng of one unique first exon (which encodes two-thirds of the lu menal domain, starting from the N-terminus, 288 amino acids long) to the f our common exons (exons 2-5, which encode the remainder of the lumenal domain, the transmembrane domain and the cytosolic tail, 246 amino acids long). Due to this unusual gene st ructure and splicing m echanism, the UGT1 isoforms have variable amino-terminal ha lves and identical carboxyl-terminal halves. While the first exon determines substrate specificity, the common exons specify the interaction with UDPGA (Ritte r et al., 1992; Gong et al., 2001). Thus, the major bilirubin UGT (UGT1A1) of humans, rats and other sp ecies is encoded by exon 1 and the adjacent 4 common exons. The phenol UGT (UGT1A6) is encoded by exon 6 and the 4 common exons. The human UGT2 gene family includes th ree members of the UGT2A subfamily and twelve members of the UGT2B subfamily (Mackenzie et al., 2005). The UGT2 proteins are encoded by separate genes consisting of six exons located on human chromosome 4q13. The region of the protein en coded by exons 1 and 2 is equivalent to that encoded by the unique exons 1 of the UGT1 isoforms, and the subsequent intron/exon boundaries are in corresponding positio ns in both gene families. Similar to

PAGE 22

9 the UGT1A enzymes, the UGT2A1 and 2A2 pr oteins have identical C-termini and different N-termini that arise due to differen tial splicing of the firs t exon (Figure 2-4). By contrast, the UGT2A3 gene comprises six exons th at are not shared with the other two. Table 2-1. Expression of huma n UGT mRNA in various tissuesa UGT Liver Intestine Esophagus & stomach Kidney Brain Prostate Other tissues 1A1 b 1A3 b 1A4 1A6 b testis, ovary 1A7 c 1A8 1A9 1A10c 2A1 Olfactory epithelium, lung 2B4 2B7 Pancreas 2B10 mammary gland, 2B11 mammary gland, adrenal, skin, adipose 2B15 mammary gland, adipose, skin, lung, testis, uterus, placenta 2B17 a Tukey and Strassburg 2000; King et al., 2000; Lin and Wong 2002; Wells et al., 2004 b only a third of the population expresses these isoforms in gastric epithelium (Strassburg et al., 1998) c expressed in bile ducts

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10 Figure 2-3. Complete human UGT1 complex locu s represented as an array of 13 linearly arranged first exons. Each first exon, except for the defective UGT1A12p and UGT1A13p pseudo ones, contains a 5´proximal TATA box el ement (bent arrow) that allows for the independent initiation of RNA polymerase activity that generates a series of overlapping RNA transcripts (A dapted from Gong et al., 2001). 2B29p 2B17p 2B15 2B10 2A3 2B27p 2B26p 2B7 2B11 2B28 2B25P 2B24P 2B4 2A1/2 5` 3` 2A1 2A2 2 3 4 5 6 Figure 2-4. The human UGT2 family. Each gene (not drawn to scale), cons isting of six exons, is represented by a white rectangle, except for ‘2A1/2’, wh ich represents seven exons (1 unique first exon and shared exons 2-6). Adap ted from Mackenzie et al. (2005). Common exons 2 3 4 5 1A12p 1A11p 1A8 1A10 1A13p 1A9 1A7 1A6 1A5 1A4 1A3 1A2p 1A1 5` 3` Exons 1 300 kb 218 kb 95 kb Primary transcripts Isozymes UGT1A1 UGT1A8 UGT1A1 UGT1A8 Etc. Etc.

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11 Sulfotransferases (SULTs) Sulfotransferases can be either membrane-bound in the Golgi or in the cytosol. While the membrane-bound SULTs sulf onate large molecules such as glucosaminylglycans, the cytosolic enzyme s are involved in the inactivation of endogenous signal molecules (steroids, thyroid hormones, neurotransmitters) and the biotransformation of xenobiotics. Each cytosolic SULT is a single / globular protein with a characteristic fivestranded parallel sheet, with -helices flanking each sheet. The active enzyme is a homodimer, with each polypeptide chain ha ving a MW of about 35,000. Kakuta et al. (1997) were the first group to solve the firs t X-ray structure for the SULT family. Mouse estrogen sulfotransferase (mEST) was show n complexed with PAP and the substrate estradiol (E2). The binding of estradiol to human SULT1A1 has also been demonstrated (Gamage et al., 2005). Both PAPSand substr ate-binding sites are lo cated deep in the hydrophobic substrate pocket. The structures of four human cytosolic enzymes have also been elucidated: SULT 1A1 (Gamage et al., 2003), dopamine/catecholamine sulfotransferase (SULT1A3) (Bidwell et al., 1999; Dajani et al., 1999), hydroxysteroid sulfotransferase (SULT2A1; hHST) (Pedersen et al., 2000), and estrogen sulfotransferase (SULT1E1; hEST) (Pedersen et al., 2002). Five SULT gene families have been identified in mammals (SULTs1-5). While SULT enzymes have different subs trate specificities, the repert oire of suitable substrates is so broad that it is not uncommon that one s ubstrate is biotransformed by more than one enzyme. SULTs are distributed in a wide variet y of tissues (Table 2-2). In humans, liver cytosol has been shown to contain mostly SULTs1A1, 2A1, and 1E1, with lesser amounts

PAGE 25

12 of SULTs 1A2, 1B1, 1E1 and 2A1. While SULT1A1 and SULT1E1 are responsible for most of the phenol and estrogen SULT he patic activity respectively, SULT2A1 (hydroxysteroid SULT) shows greater affinity for alcohols and benzylic alcohols (Mulder and Jakoby, 1990; Glatt, 2002). Table 2-2. Tissue distribution of SULTs (cDNA and mRNA) in humansa SULT Liver Intestine Esophagus & stomach Kidney Brain Lung Other tissues 1A1 Platelets 1A2 1A3 Platelets 1B1 Spleen, kidney, leukocytes 1C2 b Ovary, spinal cord, hearta 1C4 b Thyroid gland, ovary 1E1 b b Endometrium, skin, mammary 2A1 Adrenal gland, ovary 2B1 c Placenta, prostate, platelets 4A1 a reviewed by Glatt 2002. b mRNA of fetal tissues c oral mucosa Using 3-hydroxy-benzo(a)pyren e (3-OH-B[a]P) and 9-OH-B[ a]P, the existence of multiple SULT isoforms in channel catfish liver and intestine, including a 3methylcholanthrene-inducible form of phe nol-SULT in liver, has been established (Gaworecki et al., 2004; James et al., 2001) . The phenol-SULT in catfish liver and

PAGE 26

13 intestine has been isolated as a 41,000 Da protein. A second protein with a molecular weight of 31,000 Da, isolated from liver, has not been identified to date. Interestingly enough, SULT activity with phenolic substrates is higher in intestine than liver (Tong and James 2000). Other hepatic SULTs isolated and characterized from fish include petromyzonol SULT from lamprey ( Petromyzon marinus ) larva (which displays 40% homology with mammalian SULT2B1a, or cholesterol SULT) and a bile steroid SULT from the shark Heterodontus portusjacksoni (Venkatachalam et al., 2004; Macrides et al., 1994).

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14 CHAPTER 3 SULFONATION OF XENOBIOTICS BY POLAR BEAR LIVER The lipophilicity and inherent chemical stability of persistent organic pollutants (POPs) renders them excellent candidates for absorption through biological membranes as well as accumulation in both organisms a nd their environment. Many POPs have been shown to biomagnify in food webs to potentially toxic levels in top predators such as the polar bear ( Ursus maritimus ), whose diet mainly consists of ringed seal ( Phoca hispida ) blubber (Kucklick et al., 2002). Since the sulfonation of xenobiotics has neve r been studied in the polar bear, the objective of this study was to investigate the e fficiency of this rout e of detoxification on a select group of known environmenta l pollutants: 4´-hydroxy-3,3´,4,5´tetrachlorobiphenyl (4´OH-PCB79), 4´ -hydroxy-2,3,3´,4,5,5´-hexachlorobiphenyl (4´OH-PCB159), 4´-hydroxy-2,3,3´,5,5´,6-hex achlorobiphenyl (4´-OH-PCB165), pentachlorophenol (PCP), tris(4-chlorophenyl)-methanol (TCPM), 2-(4-methoxyphenyl)2-(4-hydroxyphenyl)-1,1,1-trichloroethan e (OHMXC), 3-hydroxybenzo(a)pyrene (3-OHB[a]P), triclosan (2,4,4´-trichloro-2´-hydroxydiph enyl ether) (Figure 3-1). The OH-PCBs were named as PCB metabolites, according to the convention suggested by Maervoet et al. (2004). Polychlorinated biphenylols (OH-PCBs), major biotransformation products of PCBs (James, 2001), have been shown to be pr esent in relatively hi gh concentrations in polar bears (Sandau and Nors trom 1998; Sandau et al., 2000). The abundance of these hydroxylated metabolites may be due to CYP induction (Letcher et al., 1996), inefficient

PAGE 28

15 Figure 3-1. Structures of sulfonation subs trates investigated in this study. (1) 3-OH-B[a]P; (2) triclosan; (3) 4 -OH-PCB79; (4) 4 -OH-PCB159; (5) 4 -OHPCB165; (6) OHMXC; (7) TCPM; (8) PCP. Full names of each compound are given in the text.

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16 Phase II detoxication, and inhibition of th eir own biotransformation. The 4´-OH-PCB79 (an oxidation product of PCB congener 77) is a potent inhibitor of the sulfonation of several substrates, including 3-OH-B[a]P in channel catfish intestine and human liver (van den Hurk et al., 2002, Wang et al., 2005), 4-nitrophenol by human SULT1A1 (Wang et al., 2006), 3,5-diiodothyronine (T2) in rat liver (Schuur et al., 1998), and estradiol by human SULT1E1 (Kester et al., 2000). Both 4´-OH-PCB159 and 4´-OH-PCB165 have been shown to inhibit the sulfonation of 3-OH-B[a]P a nd 4-nitrophenol by human SULT (Wang et al., 2005, 2006). Another compound detect ed in polar bears is PCP (Sandau and Norstrom 1998), a commonly used wood preserva tive that has been implicated in thyroid hormone disruption in Arctic Inuit populations (Sandau et al., 2002). TCPM is a globally distributed organochlorine co mpound of uncertain origin, which was reported in human adipose tissue (Minh et al., 2000). Polar be ar liver contains 4000-6800 ng/g lipid weight TCPM, the highest levels record ed for this compound in all species studie d (Jarman et al., 1992). TCPM is a potent androgen receptor antagonist in vitro (Schrader and Cooke 2002). OHMXC, formed by demethylation of th e organochlorine pesticide methoxychlor, is an estrogen receptor (ER) agonist, an ER antagonist and an androgen receptor antagonist (Gaido et al., 2000). The ubiquitous environmental pollutant benzo[a]pyrene is mainly metabolized to 3-OH-B[a]P, a pr ocarcinogen that can be eliminated via sulfonation (Tong and James 2000). Together with its 7,8-dihydrodiol-9,10-oxide and 7,8-oxide metabolites, 3-OH-B[a]P can form ad ducts with macromolecules and initiate carcinogenesis (Ribeiro et al., 1986). Triclosan is an an timicrobial agent that has been detected in human plasma and breast milk (Adolfsson-Erici et al., 2002). In vitro studies

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17 have shown that triclosan inhibits various biotransformation enzymes, including SULT and UDP-glucuronosyltransferases (UGT) (Wang et al., 2004). The fact that 3-OH-B[a]P, triclosan, OHMXC, 4´-OH-PCB79, 4´-OH-PCB159 and 4´-OH-PCB165 have not been re ported as environmental contam inants in polar bears to date may be due to non-significant levels in the Arctic environment or efficient metabolism via, for example, sulfonation. On the other hand, the pr esence of PCP and, particularly, high amounts of TCPM in th ese Arctic carnivores, may indicate poor sulfonation of these substrates. The polyc hlorobiphenylols 4´OH-PCB159 and 4´-OHPCB165 are of interest since t hough they have not been detected in polar bears, they are structurally similar to 4´-OH-PCB172, one of the majo r OH-PCBs found in polar bear plasma (Sandau et al., 2000). It is thus possi ble that these compounds are sulfonated with similar efficiencies. The other major Phase II biotransformation pathway for the abovementioned compounds is glucuronidation. Polar bear liver efficien tly glucuronidated 3OH-B[a]P and several OH-PCBs (Sacco and James 2004). Hypothesis Sulfonation occurring in polar bear liver is an inefficient route of detoxification for a structurally diverse group of environmental contaminants. Methodology Unlabeled PAPS was purchased from the Dayton Research Institute (Dayton, OH). Uridine 5’-diphosphoglucuronic acid (UDPGA ) was obtained from Sigma (St.Louis, MO). Radiolabeled [35S]PAPS (1.82 or 3.56 Ci/mmol) wa s obtained from Perkin-Elmer Life Sciences, Inc. (Boston, MA). The benz o[a]pyrene metabolites 3-OH-B[a]P, B[a]P-3O-sulfate and B[a]P-3-O-glucuronide were supplied by the Midwest Research Institute (Kansas City, MO), through contact with th e Chemical Carcinogen Reference Standard

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18 Repository of the National Cancer Institut e. Dr. L.W.Roberts on, U of Iowa, kindly donated the 4´-OH-PCB79, and 4´-OH-PCB1 59 and 4´-OH-PCB165 were purchased from AccuStandard, Inc. (New Haven, CT). PCP from Fluka Chemic al (Milwaukee, WI) was used to prepare the water-soluble sodi um salt (Meerman et al., 1983). Triclosan and sulfatase (Type VI from Aerobacter , S1629) were purchased from Sigma (St.Louis, MO), while methoxychlor and TCPM were purchased from ICN Biomedical (Aurora, OH) and Lancaster Synthesis, Inc. (Pelham, NH), respectively. The OHMXC was prepared by the demethylation of methoxychlor and purified by recrystalliz ation (Hu and Kupfer 2002). Tetrabutyl ammonium hydrogen sulfate (PIC -A low UV reagent) was from Waters Corporation, Milford, MA. Other reagents were the highest grade available from Fisher Scientific (Atlanta, GA) and Sigma. Animals. The samples used in this study were a kind donation from Dr. S. Bandiera (U British Columbia) and Dr. R. Letcher (Environment Canada). They were derived from the distal portion of the right lobe of livers of three adult ma le bears G, K and X. Bears G and K were collected as part of a legally-controlled hunt by I nuit in the Canadian Arctic in April 1993 near Resolute Bay, Northwest Territories, while bear X was collected in November 1993 near Churchill, Manitoba, ju st after the fasting period. Liver samples were removed within 10-15 minutes after d eath, cut into small pieces and frozen at 196ºC in liquid N2. The samples were subseque ntly stored at -80ºC. Cytosol and Microsomes Preparation. Prior to homogenization, the frozen polar bear liver samples (~2g) were gradually th awed in a few ml of homogenizing buffer. Homogenizing buffer consisted of 1.15% KCl, 0.05 M K3PO4 pH 7.4, and 0.2 mM phenylmethylsulfonyl fluoride, added from concen trated ethanol solution just before use.

PAGE 32

19 Resuspension buffer consisted of 0.25 M sucrose, 0.01 M Hepes p H 7.4, 5% glycerol, 0.1 mM dithiothreitol, 0.1 mM ethylene diamine tetra-acetic acid and 0.1 mM phenylmethyl sulfonyl fluoride. The liver was placed in a volume of fresh ice-cold buffer equal to 4 times the weight of the liver sample. The cyto sol and microsomal fractions were obtained using a procedure described previously (W ang et al., 2004). Microsomal and cytosolic protein contents were measured by the Lowr y assay, using bovine serum albumin (BSA) as standard. Sulfotransferase Assays A. Fluorometric method. The activity was measured on the basis that at alkaline pH, the benzo[a]pyrene-3-O-sulfate has different wavelength optima for fluorescence excitation and emission (294/415 nm) from the benzo[a] pyrene-3-O-phenolate anion (390/545 nm) (James et al., 1997). Saturating concentrations of PAPS were determined by performing the assay at 1 M 3-OH-B[a]P. The reaction mixture fo r detecting the sulfation of 3-OHBaP by polar bear liver cytosol c onsisted of 0.1 M Tris-Cl buffer ( p H 7.6), 0.4% BSA, PAPS (0.02 mM), 25 g polar bear hepatic cytosolic protein, and 3-OH-B[a]P (0.05-25 M) in a total reaction volume of 1.0 mL. SULT activity (pmol/min/mg) was calculated from a standard curve prepared with B[a]P3-O-sulfate standards. Substrate consumption did not exceed 10%. B. Radiochemical extraction method. This method, based on Wang and co-workers (2004), was employed in the study of the sulfonation of 4´-OH-PCB79, 4´-OH-PCB159, 4´-OH-PCB165, triclosan, PCP, TCPM and OHM XC. Cytosolic protein concentrations and incubation time were optimized for every test substrate to ensure that the reaction was linear during the incubation period. Subs trate consumption did not exceed 5%. The

PAGE 33

20 incubation mixture consisted of 0.1 M Tris-Cl buffer ( p H 7.0), 0.4% BSA in water, 20 M PAPS (10% labelled with 35S), 0.1 mg polar bear hepatic cytosolic protein, or 0.005 mg in the case of 4´-OH-PCB79 and triclosan, and substrate in a total reaction volume of 0.1 mL, or 0.5 mL in the case of TCPM. The OH-PCBs, triclosan and OHMXC were added to tubes from methanol solutions , and the methanol was removed under N2 prior to addition of other components. The TCPM was dissolved in DMSO, the solvent being present at a concentration not exceeding 1% in the final assay volume. Control determinations utilizing 1% DMSO had no inhibitory effect on sulfonation. Aqueous solutions of sodium pentachlorophenolate we re utilized in the case of PCP. Tubes containing all components except the co-substrate were placed in a water bath at 37ºC and PAPS was added to initiate the reacti on. Incubation times were 5 min (TCPM), 20 min (4´-OH-PCB79, triclosan), 30 min (PCP ) and 40 min (OHMXC, 4´-OH-PCB159, 4´OH-PCB165). The incubation was terminated by the addition of an e qual volume of a 1:1 mixture of 2.5% acetic acid and PIC-A and wa ter. The sulfated product was extracted with 3.0 mL ethyl acetate as described prev iously (Wang et al., 2004) and the phases were separated by centrifugation. Duplicate portions of th e ethyl acetate phase were counted for quantitation of sulfate conjugates. C. Radiochemical TLC method. Since the ethyl acetate phase contains sulfate conjugates formed from both the substrate of interest and substrates already present in polar bear liver, TLC was used to quantify substrate sulfation in cases where SULT activity was similar in samples and substrate blanks. After evapor ating 2 ml of ethyl acetate extract from the SULT assay under N2, the solutes were reconstituted in 40 L methanol. For 4´-OH-PCB159, 4´-OH-PC B165, PCP and OHMXC, the substrate

PAGE 34

21 conjugates were separated on RP-18F254s reverse phase TLC pl ates with fluorescent indicator (Merck, Darmstadt, Germany) us ing methanol:water (80:20). For TCPM, Whatman KC18F reverse phase 200 m TLC plates with fluorescent indicator in conjunction with a developing solvent system consisting of methanol:water:0.28 M PICA (40:60:1.9 by volume) were employed. Electr onic autoradiography (Packard Instant Imager, Meriden, CT) was used to identify a nd quantify the radioactive bands separated on the TLC plate. The counts representing the substrate sulfate conjugate products were expressed as a fraction of the total radioact ivity determined by scintillation counting, thus enabling the radioactivity due to the substr ate conjugate to be accurately determined. The identity of the conjugate of TCPM as a sulfate ester was verified by studying its sensitivity to sulfatase. Polar bear cytoso l (0.5 mg) was incubated for 75 minutes with or without 200 µM TCPM. The incubation was terminated, and the product extracted into ethyl acetate as above. The et hyl acetate was evaporated to dryness and dissolved in 0.25 mL of Tris buffer, p H 7.5, containing 0 or 0.08 units of sulfatase. Followi ng an overnight incubation at 35 û C, the reaction was stopped by the a ddition of methanol and the tubes were centrifuged. The supernatants were evaporated to dryness, reconstituted in methanol and analyzed by TLC as described above. UDP-Glucuronosyltransferase Assay. The reaction mixture for detecting the glucuronidation of 3-OH-B[a]P by polar bear liver microsomes consisted of 0.1 M TrisHCl buffer ( p H 7.6), 5 mM MgCl2, 0.5% Brij-58, UDPGA (4 mM), 5 g polar bear hepatic microsomal protein, and 3-OH-B[ a]P in a total reaction volume of 500 L. The substrate, 3-OH-B[a]P in me thanol, was blown dry under N2 in the dark in a tube to which, after complete evaporation, a premixed solution of microsomal protein and Brij-

PAGE 35

22 58 (in a 5:1 ratio) was added, vortexed, and le ft for 30 minutes on ice. Subsequently, the buffer and water were added in that order and vortex-mixed. Immediately preceding a 20minute incubation at 37ºC, UDPGA was added to initiate the reac tion. The reaction was terminated by the addition of 2 mL ice-cold methanol. Preci pitated protein was pelleted by centrifugation at 2000 rpm for 10 minutes. The supernatant, 2 mL, was then mixed with 0.5 mL NaOH (1N) and the fluorescence of B[a]P-3-glucuronic acid measured at excitation/emission wavelengths of 300/421 nm (Singh & Wiebel, 1979). The activity of UGT (nmol/min/mg) was then determined. Preliminary studies established the conditions for linearity of reaction with respect to time, protein and detergent concentrations , at the same time ensuring that substrate consumption did not exceed 10%. The apparent Km for UDPGA was determined by performing experiments at a fixed concentration of 3-OH-B[a]P (10 M). Saturating UDPGA concentrations were used in order to determine 3-OH-B[a]P glucuronidation kinetics. Kinetic Analysis. Duplicate values for the rate of c onjugate formation at each substrate concentration were used to calculate kinetic parameters using Prism v 4.0 (GraphPad Software, Inc., San Diego, CA). Equations used to fit the data were the Michaelis-Menten hyperbola for one-site binding (eq. 1), the Hill plot (eq. 2), substrate inhibition for onesite binding (eq. 3) (H ouston and Kenworthy 2000), and part ial substrate inhibition due to binding at an allosteric site (eq. 4) (Zhang et al., 1998). v = Vmax[S] / (Km + [S]) (1) v = Vmax[S]h / (S50 h + [S]h) (2) v = Vmax[S] / (Km + [S] + ([S]2/Ki)) (3)

PAGE 36

23 v = Vmax1(1 + (Vmax2[S]/Vmax1Ki)) / (1 + Km/[S] + [S]/Ki) (4) Values for Km and Vmax derived from equation 1 were us ed as initial values in the fitting of data to equations 3 and 4. Eadie-Hofs tee plots were used in order to analyze the biphasic kinetics observed. Results Sulfonation and glucuronidation of 3-OH-B[a]P Optimum conditions for sulfonation were 10 minutes incubation time and 25 µg cytosolic protein. A concentration of 0.02 mM PAPS provided satura ting concentrations of the co-substrate and enabled kinetic parame ters at 1.0 µM 3-OH-B[a]P to be calculated by the application of eq. 1 (Table 3-1a). Th e data for the sulfonation of 3-OH-B[a]P was fit to a two-substrate model (eq. 3), whereby the binding of a second substrate to the enzyme is responsible for the steep declin e in enzyme activity at concentrations exceeding 1 µM (Figure 3-2a). Initial estimates of Vmax1 and Km were provided by the initial data obtained at low [S] (non-inhibitory), while Vmax2 was constrained to 65 20 pmol/min/mg, which is slightly be low the plateau in Figure 3-2a. The kinetic scheme (Figure 3-2b) illu strates the proposed partial substrate inhibition process, which assumes that substr ate binding is at equilibrium, which is probable due to the low turnover rate of SULT. The best fit of the data was provided by a Ki of 1.0 0.1 µM. Binding of the second substrate molecule results in a tenfold reduction in the rate of sulfonate formation.

PAGE 37

24 Table 3-1. Estimated kinetic parameters (Mean SD) for (a) sulfonation and (b) glucuroni dation of 3-OH-B[a]P by polar bear liver cytosol and microsomes. Values were cal culated as described in the Methodology. (a) sulfonation Substrate Vmax1 (app) Km (app) Vmax1/Km Vmax2 (app) a Ki (app) Vmax2/Ki (pmol/min/mg) (µM) (µL/min/mg) (pmol/min/mg) (µM) (µL/min/mg) 3-OH-B[a]P 500 8 0.41 0.03 1220 70 65.0 20.0 1.01 0.10 66.2 26.8 PAPS 162 35 0.22 0.07 ----(b) glucuronidation Substrate Vmax (app) Km (app) Vmax / Km (nmol/min/mg) (µM) (µL/min/mg) 3-OH-B[a]P 3.00 1.18 1.4 0.2 1900 544 UDPGA 1.53 0.56b, 1.47 0.48c 42.9 2.5b, 200 68c -a constrained variable s to obtain best fit b values for high-affinity component c values for low-affinity component

PAGE 38

25 Figure 3-2. Sulfonation of 3-OHB[a]P at PAPS = 0.02 mM. A. Each data point represents the average of duplicate assays for each bear, while the error bars represent the standa rd deviation. The line represents the best fit to the data of equation (3). B) Kinetic model for partial substrate inhibition of SULT by 3-OH-B[a]P, afte r Zhang et al. (1998). E refers to SULT.

PAGE 39

26 Optimum conditions for the glucuroni dation of 3-OH-B[a]P by polar bear microsomes were found to be 5 µg micros omal protein and a 20-minute incubation. A concentration of 4 mM UDPGA was determined to be suitable for providing saturating concentrations of the co-substrate. The binding of UDPGA to UGT at 10 µM 3-OHB[a]P was shown to be biphasic, with a fi vefold reduction in affinity at higher UDPGA concentrations (Table 3-1b). The kinetic para meters for the co-substrate were calculated by deconvoluting the curvilinear data in the Eadie-Hofstee plot (Figure 3-3). In the presence of 4 mM UDPGA, the formation of B[a]P-3-O-glucuronide followed MichaelisMenten kinetics (Table 3-1b). Figure 3-3. Eadie-Hofstee plot for the glucuronidation of 10 µM 3-OH-B[a]P, over a UDPGA concentration ra nge of 5-3000 µM. Each data point represents the average of duplicate assays for all bears, while the error bars represent the standard deviation.

PAGE 40

27 Sulfonation of other substrates Triclosan sulfate was formed rapidly, w ith the overall kinetics conforming to a hyperbolic curve (eq. 1) (Table 3-2). Substr ate inhibition was obser ved for 4´-OH-PCB79 (Figure 3-4), with the data fitting equation (3). The value of Ki that gave the best fit was 217 25 µM (Table 3-2). Sulfate conjugati on of 4´-OH-PCB159 and 4´-OH-PCB165, which proceeded via Michaelis-Menten kineti cs, was, respectively, 11 and 5 times less efficient than the sulfonation of 4´-OH-PCB 79 (Table 3-2). At a concentration of 10 µM, 4´-OH-PCB165 was observed to in hibit sulfonation of substrates already present in polar bear liver cytosol by 60%. Table 3-2. Kinetic parameters (Mean SD) for the sulfonation of various xenobiotics by polar bear liver cytosol, listed in order of decreasing enzymatic efficiency. All data fit equation (1), except fo r 4´-OH-PCB79 and PCP, which fit equations (3) and (2) respectively (see Methodology for equations). Substrate Vmax Km Vmax / Km Ki (pmol/min/mg) (µM) (µL/min/mg) (µM) ________________________________________________________________________ triclosan 1008 135 11 2 90.8 6.8 4´-OH-PCB79 372 38 123 20 3.1 0.3 217 25a OHMXC 51.1 7.8 67 4 0.8 0.1 4´-OH-PCB165 8.6 2.0 17 7 0.56 0.17 TCPM 62.0 11.2 144 36 0.44 0.06 4´-OH-PCB159 14.8 2.3 60 21 0.28 0.12 PCP 13.8 1.2 72 14b 0.20 0.05 aKi for bears G, K and X were 240, 220 and 190 µM respectively. These values were constrained to obtain the best fit for the data bS50; h = 2.0 0.4

PAGE 41

28 Figure 3-4. Sulfonation of 4´ -OH-PCB79, PAPS = 0.02 mM. Each data point represents the average of duplicate assays for each bear, while the error bars represent the standard de viation. The line repres ents the best fit to equation (4) for 4´-OH-PCB79. Due to variable rates of sulfonation of these unknown substrates, autoradiographic counts corresponding to the OHMXCO -sulfate band were used to correct the activities calculated from the scintillation counter data (Figure 3-5). This enabled the transformed data to be fit into a Michaelis-Menten mode l (Table 3-2). The autoradiograms obtained showed that increasing concen trations of OHMXC resulted in decreased counts for the unknown sulfate conjugates (Figure 3-5). Sulf onation of the unknown substrates in polar bear cytosol was reduced by half at OHMXC concentrations < 20 µM.

PAGE 42

29 Figure 3-5. Autoradiogram showing the re verse-phase TLC sepa ration of sulfonation products of OHMXC. Incubations were carried out with the indicated concentrations of OHMXC. The arrow indicates the sulfate conjuga te of the OHMXC, while other bands represent unidentified su lfate conjugates formed from endobiotics or other xenobiotics in polar bear liver cytosol. The total TCPM sulfate conjugate producti on formed after 5 minutes under initial rate conditions did not exceed 30 pmol. TLC, followed by autoradiography, was thus used to distinguish the TCPM-sulfate band (Rf 0.54) from other sulfate conjugates (Rf 0.05 and 0.72) originating from compounds in th e polar bear liver cytosol (Figure 3-6). The data obtained followed hyperbolic ki netics (Table 3-2). Even though the TLC from the kinetic experiments showed a TCPM concentration-dependent increase of the band corresponding to the purported TCPM-sulfa te, and this band was absent in the

PAGE 43

30 substrate blank, the fact remained that we were apparently looking at the only instance ever reported of a successful sulfonati on of an acyclic tertiary alcohol. Figure 3-6. Autoradiogram showing the re verse-phase TLC sepa ration of sulfonation products from incubations with TCPM using polar bear (P), channel catfish (C), and human (H) liver cytosol in the absence of (0), a nd presence of 100 µM TCPM (100). The arrow indicates the sulfate conjugate of the substrate, while other bands represent unidentified su lfate conjugates formed from endobiotics or other xenobiotics in liv er cytosol. Thus, additional experiments were perfor med to verify the identity of this conjugate. The purity of the TCPM was tested in the event that the additional band was due to an impurity in the substrate. However, the substrate used was found to be free of contaminants by HPLC (C18 reverse phase co lumn, with detection at 268 and 220 nm, using 90% methanol in water and a flow rate of 1 mL/min). A single peak was recorded

PAGE 44

31 at 7.3 minutes. Another experiment involved a 60-minute incubation performed with 100 µM TCPM and 0.1 mg cytosolic protein from polar bear, channel catfish and human liver. For each of the three species, we detected a conjugate at Rf = 0.54. The substrate blanks showed no band at the same position (Figure 3-6). The TCPM sulfate conjugate from polar bear could be hydr olyzed by sulfatase (Figure 37), providing further evidence of the sulfonation of this alcohol. Figure 3-7. Autoradiogram showing the re verse-phase TLC sepa ration of sulfonation products of TCPM and the effect of sulfatase treatment. A, incubation in the absence of TCPM (lane 1), and following treatment with sulfatase (lane 2). B, incubation with 200 µM TCPM (lane 3), and following treatment with sulfatase (lane 4). The a rrow indicates the su lfate conjugate of the TCPM, while other bands represent unidentified sulfate conjugates formed from endobiotics or other xenobiotic s in polar bear liver cytosol. Inhibition of sulfonation of substrates alr eady present in the polar bear liver was noted upon adding 1 µM PCP (Figure 3-8). The data for PCP sulfonation fitted the nonlinear Hill plot (eq. 2) (Table 3-2).

PAGE 45

32 Figure 3-8. Autoradiogram showing revers e-phase TLC separation of sulfonation products from the study of PCP kinetics. The arrow indicates the sulfate conjugate of PCP, while other bands represent unidentified sulfate conjugates formed from endobiotics or other xenobiotics in polar bear liver cytosol. Discussion The sulfonation of hydroxylated metabolites of benzo[a]pyrene has been reported in various species, including fish (James et al., 2001) and humans (Wang et al., 2004). Benzo[a]pyrene-3-glucuronide has been show n to be produced by fish (James et al., 1997), rats (Lilienblum et al ., 1987) and humans (Wang et al., 2004). There are, however, few studies investigating the ki netics of these conj ugation reactions. Glucuronidation of 3-OH-B[a]P was more efficien t in polar bear liver than in human liver or catfish intestine. On the other hand, the efficiency of su lfonation was similar to that shown in human liver but around three times less than in catfish intestine (Wang et al.,

PAGE 46

33 2004, James et al., 2001). From the limited comparat ive data available, it can be surmised that, in general, polar bear liver is an important site of 3-OH-B[a]P detoxication, particularly with respect to glucuronidation. Substrate inhibition for the sulfonation of 3-OH-B[a]P has been observed at relatively low concentrations of the xenobiotic in other species such as catfish and human (Tong and James 2000, Wang et al., 2005). Data from the polar bear sulfonation assay fitted a two-substrate model developed for the sulfonation of 17 -estradiol by SULT1E1 (Zhang et al., 1998). This model was also used to explain the sulf onation profile observed for the biotransformation of 1-hydroxypyrene, a compound structurally similar to 3-OHB[a]P, by SULTs 1A1 and 1A3 (Ma et al., 200 3). In the original model, SULT1E1 was saturated with PAPS, and each of the estrad iol substrate molecules bound independently to the enzyme. The estradiol binding sites were proposed to consist of a catalytic site, and an allosteric site that re gulates turnover of the substr ate (Zhang et al., 1998). The substrate inhibition observed with polar bear liver cytosol at higher 3-OH-B[a]P concentrations (>0.75 µM) can thus be expl ained by the binding of a second substrate molecule to an allosteric site, which leads to a two-fold decrease in affinity and an eightfold decrease in Vmax. SULTs are generally high-affinity, low-cap acity biotransformation enzymes that operate effectively at low substrat e concentrations. Thus, typical Kms for the sulfonation of xenobiotic substrates are us ually significantly lower than Kms for the same substrates undergoing biotransformation by low-affinity , high-capacity glucur onosyltransferases (UGTs). In polar bear liver, both pathways s howed similar apparent affinities for 3-OHB[a]P, with Kms of 0.4 and 1.4 µM for sulfonation and glucuronidation respectively,

PAGE 47

34 suggesting these two pathways of Phase II metabolism compete at similar 3-OH-B[a]P concentrations. However, the apparent maximal rate of sulfonation was about 7.5 times lower than the rate of glucuronidation. It was previously reported that the ma ximum rate of gluc uronidation of 3-OHB[a]P by polar bear liver was 1.26 nmol/min/mg, or around half the Vmax value obtained in this study (Sacco and James 2004). However, the preceding study utilized 0.2 mM UDPGA, which, as seen from Table 3-2a, is equivalent to the Km (for UDPGA) of the low-affinity enzyme, and thus does not repr esent saturating concentrations of the cosubstrate. The affinity of the enzyme for 3-OH-B[a]P did not change significantly with a 20-fold increase in UDPGA concentrations , suggesting that substrate binding is independent of the binding of co-substrat e. The binding of UDPGA was biphasic, indicating that multiple hepatic UGTs may be responsible for the biotransformation. Biphasic UDPGA kinetics have also been demonstrated in human liver and kidney for 1naphthol, morphine, and 4-methylumbelliferone (Miners et al., 1988a,b; Tsoutsikos et al., 2004). While Vmax was similar for both components, there was a fivefold decrease in enzyme affinity for UDPGA as the co-subs trate concentration was increased. The involvement of at least two enzymes can be physiologically advantage ous since it enables the maintenance of a high turnover rate even as UDPGA is consumed. Although physiological UDPGA concentrations in pol ar bear liver are unknown, mammalian hepatic UDPGA has been determined to be around 200-400 µM (Zhivkov et al., 1975, Cappiello et al., 1991), implyi ng that the observed nonlinear ki netics in the polar bear may operate in vivo .

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35 The rate of triclosan sulfonation was the highest of all the substrates studied; apparent Vmax was twice as high as fo r 3-OH-B[a]P. However, th e overall efficiency of sulfonation of the hydroxylated PAH was s till 13 times higher than for triclosan sulfonation. The presence of three chlorine substituents (though none flanking the phenol group) does not hinder the sulfonation of triclo san when compared to the ‘chlorine-free’ 3-OH-B[a]P. Triclosan sulfonation in polar be ar liver was similar to human liver with respect to enzyme affinity; however the maxi mum rate was tenfold higher in polar bears than in humans (Wang et al., 2004). This may be one reason why triclosan has not been detected in polar bear plasma or liver to date. Our data fitted a model that indicates the substrate inhibition observed for 4´-OHPCB79 may be due to a second substrate mol ecule interacting with the enzyme-substrate complex at the active site rather than an a llosteric site, resulting in a dead-end complex. Unlike 3-OH-B[a]P, sulfonation can only proceed via the single substrate-SULT complex. Models of SULT1A1 a nd 1A3, with two molecules of p -nitrophenol or dopamine at the active site respectively, have been proposed as a mechanism of substrate inhibition (Gamage et al., 2003, Ba rnett et al., 2004), while the crystal structure of human EST containing bound 4,4´-OH-3,3´ ,5,5´-tetrachlorobiphenyl at the active site has not provided any evidence of an allosteric site (Shevtsov et al., 2003). The slower sulfonation of 4´-OH-PCB79 compared with 3-OH-B[a]P may result from the inductive effect of the chlorines flanking the phenolic group rather than steric hindrance (Duffel and Jakoby, 1981). However, polar bear liver sulfonated 4´-OH-PCB79 more rapidly than the other OH-PCB substrates studied.

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36 The inclusion of two additional chlorine substituents on the non-phenol ring (with respect to 4´-OH-PCB79) re sulted in both 4´-OH-PCB1 59 and 4´-OH-PCB165 being very poor substrates. Inefficient sulfona tion may be one reason why the related compound 4´-OH-PCB172 accumulates in pola r bears. Some degree of substrate inhibition may also be expected to contribute to this accu mulation, as was observed with 4´-OH-PCB165. Sulfonation was not an efficient pathway of OHMXC detoxification. The rate of OHMXC-sulfonate formation was around 7 times lower than for 4´-OH-PCB79. Since resonance delocalization of negative char ge on the phenolic oxygen by the flanking chlorines in chlorophenols may decrease Vmax by increasing the energy of the transition state of the reaction (Duffel and Jakoby, 1981), it is possible that in the case of OHMXC (with no chlorines flanking the phenolic grou p), product release, ra ther than sulfonate transfer, may have been the rate-limiting step. TCPM was a poor substrate for sulfonation, and this may be one reason why it has been measured in such high amounts in polar bear liver. To our knowledge, sulfonation of acyclic tertiary alcohols has not been reported in the literature. Despite the considerable steric hindrance of thr ee phenyl groups, the alcohol group could be sulfonated. Although the al cohol in TCPM is not of the be nzylic type, the presence of three proximal phenyl groups ma y give this group some benz ylic character, rendering sulfonation of the alcohol possible. Both SU LT 1E1 and SULT 2A1 have been shown to sulfonate benzylic alcohol groups attached to large molecule s (Glatt, 2000). Sulfation of the benzylic hydroxyl group leads to an unstable sulfate conjuga te that readily degrades to the reactive carbocation or spontaneously hydrolyzes back to the alcohol. Attempts to

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37 recover TCPM-O-sulfonate from TLC plates resulted in recovery of TCPM from the conjugate band, perhaps because of the conjugate’s instability. A study of the sulfonation of PCP was compli cated by the fact that it is a known SULT inhibitor, often with Kis in the submicromolar range. In our experiments, this was seen as a 74% decrease in formation of the unidentified sulfonate conjugates (band shown at the solvent front in Figure 3-8) upon addition of 1 µM PCP. Although PCP was a strong inhibitor of SULT1E1 (Kester et al., 2000), and has been postulated to be a deadend inhibitor for phenol sulfotransferases (Duffel and Jakoby, 1981), it was possible that polar bear SULT 1A isoforms were not comple tely inhibited by PCP, or that other SULT isoform(s) were responsible for the limited sulfonation activity observed. Thus, we have shown that, in vitro at least, one mammalian species is capable of limited PCP sulfonation. Even though the tertiary alc ohol of TCPM was a poor candidate for sulfonation, it was metabolized at twice the efficiency of PCP, which has a phenolic group that is usually more sus ceptible to sulfonation. This demonstrates the extent of the decreased nucleophilicity on the phenolic oxyg en due to the resonance delocalization afforded by the five chlorine substituents. Conclusions In summary, this study demonstrated that , in polar bear liver, 3-OH-B[a]P was a good substrate for sulfonation an d glucuronidation. Other, ch lorinated, substrates were biotransformed with less efficiency, impl ying that reduced rate s of sulfonation may contribute to the persistence of compounds such as hexachlori nated OH-PCBs, TCPM and PCP in polar bear tissues.

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38 CHAPTER 4 GLUCURONIDATION OF POLYCHLORINATED BIPHENYLOLS BY CHANNEL CATFISH LIVER AND INTESTINE Polychlorinated biphenyls (PCBs) were extens ively used as dielectrics in the midtwentieth century. Despite a ban on their use in the US, Europe and Japan since the mid 1970s, the chemical stability of PCBs has resulted in their persistence at all trophic levels around the globe. Enzyme-mediated biotransform ation is an important influence on PCB persistence, and its significance in PCB toxi cokinetics is dependent on congener structure and the metabolic capacity of the organism. Polychlorinated biphenylols (OH-PCBs) ar e products of CYP-dependent oxidation of PCBs (James 2001). While OH-PCBs are more polar than their parent molecules, they are still lipophilic enough to be orally absorbed, and dist ribute to several tissues (Sinjari et al., 1998). Thus, not only have these compo unds been detected in the plasma (which represents recent dietary exposure, biotra nsformation, and remobilization into the circulation) of a variety of animal species , such as polar bear (Sandau et al., 2004), bowhead whale (Hoekstra et al., 2003), catfish (Li et al., 2003), a nd humans (Fängström et al., 2002; Hovander et al., 2002), but al so significantly, from a developmental toxicology aspect, in fetuses and breast milk (Sandau et al., 2002; Guve nius et al., 2003). OH-PCBs may contribute signi ficantly to the recognized t oxic effects of PCBs such as endocrine disruption (Safe 2001; Shirai shi et al., 2003), tumor promotion (Vondrá ek et al., 2005) and neurological dysfunction (Sha rma and Kodavanti 2002; Meerts et al., 2004).

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39 Elimination of these toxic metabolites vi a Phase II conjugation reactions, such as glucuronidation and sulfonation, are thus importa nt routes of detoxification. In view of the persistence of certain OH-PCBs, it is surprising that only a few studies have attempted to investigate the biotransformati on of these compounds in animals or humans, particularly by glucuronidation (Tampal et al., 2002; Sacco and James 2004; Daidoji et al., 2005), which is normally a highe r-capacity pathway than sulfonation. Glucuronidation is catalyzed by a family of endoplasmic reticular membrane-bound enzymes, the UDP-glucuronosyltransferases (UGTs), which transfer a D-glucuronic acid moiety from the co-substrate UDP-glucuronic acid (UDPGA) to a xenobiotic containing a suitable nucleophilic atom such as oxygen, nitrogen and sulfur. UGTs are mainly found in the liver, but also in extrahepatic tissues , such as the small intestine and kidney (Wells et al., 2004). The various chlorine and hydroxyl substi tution patterns possible on the biphenyl structure may lead to significant differences in glucuronidation kine tics. One explanation for the retention of certain OH-PCBs may t hus be that they are poor substrates for glucuronidation. Tampal and co -workers (2002) studied the gl ucuronidation of a series of OH-PCBs by rat liver microsomes. Efficiency of glucuronidation varied widely, and substitution of chlorine atoms at the m and p -positions on the nonphenolic ring greatly lowered Vmax. Weak relationships were observe d between the dihedral angle, p Ka, log D and enzyme activity. The experimentally determ ined kinetic parameters determined in the Tampal et al study were subsequently rela ted to the physicochemical properties and structural features of the OH-PCBs by means of a quanti tative structure-activity

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40 relationship (QSAR) study. Hydr ophobic and electronic aspect s of OH-PCBs were shown to be important in their glucuronidation (Wang, 2005). Most of the persistent OH-PCBs found in human plasma are hydroxylated at the p position, in addition to being meta -chlorinated on either side of the phenolic group. The remaining substitution pattern on both rings is highly variable (Bergman et al., 1994; Sjödin et al., 2000). An OH group in the para position, with two flanking chlorine atoms was associated with estrogen and thyroid hor mone sulfotransferase inhibitory activity (Kester et al., 2000; Schuur et al., 1998), a nd exhibited the highest affinity for transthyretin (TTR) (Lans et al., 1993), th e major transport protein in non-mammalian species (Cheek et al., 1999). Such OH-PCBs we re potent inhibitors of the sulfonation of 3-hydroxybenzo[a]pyrene (Wang et al., 2005). In contrast, the OH-PCBs having an unhindered hydroxyl group substituted at the para position (relative to the biphenyl bond) have exhibited the strongest binding to th e rodent estrogen receptor (ER), although the competitive ER binding affinities were 100-fold lower than that observed for estradiol (Korach et al., 1988; Arulmo zhiraja et al., 2005). In the channel catfish, individual OH-P CBs have been shown to inhibit the in vitro intestinal glucuronidation of several hydroxylated metabolites of benzo[a]pyrene (BaP) (van der Hurk et al., 2002; Ja mes and Rowland-Faux 2003). The in situ hepatic glucuronidation of a procar cinogenic BaP metabolite, the (-)benzo[a]pyrene-7,8-dihydrodiol, was also inhibited by a mixture of OH-PCBs, conseque ntly increasing the formation of DNA adducts (James et al., 2004). It is possibl e that these compounds inhibit their own glucuronidation. The OH-PCB metabolites of 3,3,4,4-tetrachlorobiphenyl (CB-77), one of the most toxic PCBs known, were poor substrates for catfish intestinal glucuronidation

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41 (James and Rowland-Faux, 2003). This may help to explain the pe rsistence of these compounds. Hypothesis The glucuronidation kinetics of a series of potentially toxic p -OH-PCBs by channel catfish liver and proximal intestine is influe nced by the structural arrangement of the chlorine substituents around the biphenyl ring. Methodology Chemicals. A total of 14 substrates were used in this study (Figure 4-1). The nomenclature of the OH-PCBs is based on the recommendations of Maervoet and coworkers (2004). The following substrates (Catalog no. in parentheses) were purchased from Accustandard (New Haven, CT): 4-OHCB2 (1003N), 4-OHCB14 (2004N), 4'-OHCB69 (4008N), 4'-OHCB72 (4009N), 4'-OHCB106 (5005N), 4'-OHCB112 (5006N), 4'OHCB121 (5007N), 4'-OHCB159 (6001N), a nd 4'-OHCB165 (6002N). The compounds 4'-OHCB35, 4-OHCB39, 4'OHCB68, 4'-OH CB79 were synthesize d by Suzuki-coupling (Lehmler and Robertson, 2001; Bauer et al., 1995). The 4-hydroxy biphenyl (4-OHBP) was purchased from Sigma (St.Louis, MO). 14C-UDPGA (196 Ci/ mol) was obtained from PerkinElmer Life and Analyt ical Sciences (Boston, MA). The 14C-UDPGA was diluted with unlabelled UDPGA to a specific activity of 1.5-5 Ci/ mol for use in enzyme assays. PIC-A (tetrabutylammoni um hydrogen sulfate) was obtained from Waters Corp. (Milford, MA). Other reagents we re the highest grade available from Fisher Scientific (Atlanta, GA) and Sigma.

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42 OH Cl Cl Cl Cl OH Cl Cl Cl Cl OH Cl Cl Cl Cl OH Cl Cl Cl Cl OH Cl Cl OH Cl Cl Cl Cl OH Cl Cl OH Cl 4`-OHCB68 4`-OHCB72 4`-OHCB69 4`-OHCB79 4-OHCB14 4-OHCB39 4`-OHCB35 4-OHCB2 OH 4-OHBP OH Cl Cl Cl 4`-OHCB121 Cl Cl OH Cl Cl Cl 4`-OHCB106 OH Cl Cl Cl Cl 4`-OHCB159 Cl Cl Cl Cl OH Cl Cl Cl 4`-OHCB112 OH Cl Cl Cl 4`-OHCB165 Cl Cl Cl Cl Cl Figure 4-1. Structure of substrates used in channel catfish glucuronidation study. Animals. Channel catfish ( Ictalurus punctatus ), with weights ranging from 2.1 – 3.7 kg, were used for this study. All fish were kept in flowing well water and fed a fish chow diet (Silvercup, Murray, UT). Care and treatment of the animals was conducted as per the guidelines of the University of Fl orida Institutional Animal Care and Use Committee. The microsomal fractions were obt ained from liver and intestinal mucosa

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43 using a procedure described previously (James et al., 1997). Only the proximal portion of the intestine was used in th e study. Protein determination wa s carried out by the method of Lowry and co-workers (1951) using bovi ne serum albumin as protein standard. Glucuronidation assay. A radiochemical ion-pair extraction method was employed to investigate the glucuronidati on of the 4-OHPCBs and 4-OHBP. Substrate consumption did not exceed 10%. Initial experiments determined the saturating concentrations of UDPGA to be employed. The incubation mixture consisted of 0.1 M Tris-Cl buffer ( p H 7.6), 5 mM MgCl2, 0.5% Brij-58, 200 M or 1500 M [14C]UDPGA (intestine and liver , respectively), 100 g catfish intestinal or he patic microsomal protein, and substrate in a total reaction volume of 0.1 mL. Initially, the OH-PCBs were added to tubes from methanol solutions and evaporated under nitrogen. In al l cases, the protein and Brij-58 were added to the dried substrate, thoroughly vort exed and left on ice for 30 minutes. Subsequently, the buffer, MgCl2, and water were added in that order and vortexmixed. After a pre-incubation of 3 minutes at 35ºC, UDPGA was added to initiate the reaction, which was terminated after 30 mi nutes incubation by the addition of a 1:1 mixture of 2.5% acetic acid and PIC-A in wa ter, such that the final volume was 0.5 mL. The glucuronide product was extracted by two successive 1.5 mL portions of ethyl acetate. The phases were separated by centrifug ation, and duplicate portions of the ethyl acetate phase were counted for quant itation of glucuronide conjugate. Physicochemical parameters. The structural characteri stics of the OH-PCBs were calculated using ChemDraw 3D (Cambridge Soft Corp., Cambridge, MA). Parameters used were: the Connolly Accessible Surface Ar ea (CAA, the locus of the center of a probe sphere, representing the solvent, as it is rolled over the molecular shape), the

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44 Connolly Molecular Surface Area (CMA, the contact surface created when a probe sphere (radius = 1.4 , the size of H2O), representing the solvent, is rolled over the molecular shape), the Connolly Solvent-Excl uded Volume (CSV, the volume contained within the contact molecular surface, or that volume of space that the probe is excluded from by collisions with the atoms of the molecule), the ovality (the ratio of the Molecular Surface Area to the Minimum Surface Area, wh ich is the surface area of a sphere having a volume equal to CSV of the molecule), and dihedral angle (the angle formed between the planes of the two rings, which is related to the extent of coplanarity of the molecule). ACD/ILab software (Advanced Chemistry De velopment, Ontario, Canada) was used to predict log P, log D (at p H 7.0), and the p Ka (of the phenolic group). Kinetic analysis. Duplicate values were employed for the rate of conjugate formation at each substrate concentration to calculate kinetic parameters using Prism v4.0 (GraphPad Software, Inc., San Diego, CA). Equations used to fit the data were the Michaelis-Menten hyperbola for one-site binding and the Hill plot for positive cooperativity. Results The kinetics for UDPGA were analyzed for the glucuronidation of three representative OH-PCBs (Table 4-1). Satura ting concentrations of UDPGA were higher in liver than in intestine (Figure 4-2). The gl ucuronidation of most of the OH-PCBs tested followed Michaelis-Menten kinetics (Figure 43A). In the case of the glucuronidation of 4`OHCB35 by liver and 4`OHCB112 by proximal inte stine, the data fitted the Hill plot (Figure 4-3B).

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45 Table 4-1. Estimated kinetic parameters (mean S.D.) for the co-substrate UDPGA in the glucuronidation of three different OH-PCBs. Substrate Substrate Vmax (app) Km (app) Concentration ( M) (nmol/min/mg) ( M) Liver 4 -OHCB-35 500 0.87 0.20 697 246 4 -OHCB-72 250 0.32 0.14 247 162 Intestine 4 -OHCB-69 200 0.20 0.11 27 14 The estimated apparent maximal rate of glucuronidation of polychlorinated biphenylols by channel catfish ranged from 124-784 pmol/min/mg for proximal intestine and 404-2838 pmol/min/mg for th e liver (Table 4-2). The Kms for individual OH-PCBs tended to be different in the two organs, with a few exceptions (4OHCB2, 4`OHCB165). Vmax was significantly higher in liver than in intestine. Conversely, the affinity of intestinal catfish UGTs (Km range: 42-572 M) for the OH-PCBs tested was higher than for liver UGTs (Km range: 111-1643 M). These contrasting diffe rences are reflected in the lack of any difference in the efficiency of glucuronidation in both organs when all the OH-PCB substrates were c onsidered (Table 4-3). Vmax for OH-PCB glucuronidation in both organs were strongly co rrelated with each other (R2=0.74). This relationship did not exist for Km (R2=0.003).

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46 A 0 250 500 750 1000 1250 1500 1750 0.00 0.25 0.50 0.75 1.00[UDPGA] ( M)v (nmol/min/mg) B 0 25 50 75 100 125 0.00 0.05 0.10 0.15 0.20 0.25 0.30[UDPGA] ( M)v (nmol/min/mg) Figure 4-2. UDPGA gluc uronidation kinetics in 4 catfish. A) in liver, using 500 M 4 -OH-CB35. B) in proximal intestine, using 200 M 4 -OH CB69

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47 A B 0 100 200 300 400 500 600 700 0 100 200 300 400 500[4`-OH CB 112] ( M)v (pmol/min/mg) Figure 4-3. Representative kinetics of the glucuronidation of OH-PCBs in 4 catfish. A) Michaelis-Menten plot for 4 -OHCB-159 by liver. B) Hill plot for 4 OHCB-112 by proximal intestine 0 250 500 750 1000 1250 1500 0 250 500 750[4`-OH CB 159] ( M)v (pmol/min/mg)

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48 Table 4-2. Kinetic parameters (Mean ± S.D.) for the gluc uronidation of 4-OHBP and OHPCBs. Intestine Liver Substrate Vmax (app) Km (app) Vmax (app) Km (app) 4-OHBP 43 ± 10 599 ± 110 182 ± 78 502 ± 235 4-OHCB2 417 ± 57 572 ± 47 2277 ± 849 583 ± 95 4-OHCB14 255 ± 59 387 ± 65 2022 ± 936 614 ± 202 4´-OHCB35 784 ± 348 265 ± 85 2838 ± 1456 455 ± 89 4-OHCB39 220 ± 90 134 ± 36 1716 ± 536 242 ± 76 4´-OHCB68 213 ± 91 119 ± 75 ND ND 4´-OHCB69 751 ± 253 42 ± 21 2774 ± 1153 1071 ± 410 4´-OHCB72 401 ± 236 183 ± 126 ND ND 4´-OHCB79 124 ± 36 87 ± 21 869 ± 318 476 ± 201 4´-OHCB106 431 ± 60 183 ± 58 1579 ± 645 798 ± 122 4´-OHCB112 401 ± 67 163 ± 24 2144 ± 1007 1643 ± 545 4´-OHCB121 220 ± 39 130 ± 21 1046 ± 408 207 ± 97 4´-OHCB159 188 ± 66 213 ± 136 681 ± 141 318 ± 91 4´-OHCB165 163 ± 26 137 ± 44 404 ± 116 111 ± 28 Units for Km and Vmax are µM and pmol/min/mg protein, respectively. Bold indicates S50 in place of Km. ND, not done. Table 4-3. Comparison of the estimated kine tic parameters for OH-PCB glucuronidation in catfish liver and proximal intestine Parameter Liver Intestine p -value Vmax (app) 1370 ± 275 364 ± 70 0.002 Km (app) 567 ± 128 210 ± 46 0.016 Vmax/Km 3.7 ± 0.6 3.4 ± 1.8 0.857 (Mean ± SEM for all OH-PCB substrates)

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49 The Vmax for glucuronidation in both proximal intestine and liver was significantly decreased upon addition of a second chlorine substituent flanking the phenolic moiety, while keeping the chlorine substitution pattern in the rest of the molecule constant (Table 4-4, Figure 4-4). The affinity of hepatic UGTs for the OH-PCBs appeared to increase with the addition of a second flanking chlori ne atom; however, this relationship did not achieve statistical significance. Table 4-4. Comparison of kinetic parameters (Mean ± SEM) for the glucuronidation of OHPCBs grouped according to the numb er of chlorine atoms flanking the phenolic group Parameter Flanking chlorines p -value 1 2 Liver Vmax (app), pmol/min/mg 2247 ± 204 1002 ± 274 0.007 Km (app), M 856 ± 209 342 ± 88 0.053 Intestine Vmax (app), pmol/min/mg 560 ± 85 190 ± 23 0.003 Km (app), M 274 ± 97 191 ± 53 0.473 The effect of chlorine substituents on th e nonphenolic ring on glucuronidation of OH-PCBs was also investigated. No significant differences on Km and Vmax could be observed between the absence or presence of specific chlorine substituents on the nonphenolic ring. The only exception was that the presence of an ortho -chlorine significantly ( p =0.03) decreased the Km in the proximal intestine.

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50 A 0 3,4 2,4,6 2,3,4,5 2,3,5,6 0 250 500 750 1000one flanking Cl two flanking Cl Substitution pattern on nonphenol ringVmax (pmol/min/mg) B 0 3,4 2,4,6 2,3,4,5 2,3,5,6 0 1000 2000 3000 4000one flanking Cl two flanking Cl Substitution pattern on nonphenol ringVmax (pmol/min/mg) Figure 4-4. Decrease in Vmax with addition of second chlorine atom flanking the phenolic group, while keeping the chlorine su bstitution pattern on the nonphenolic ring constant. A) proximal intestine. B) liver.

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51 Regression analysis was performed be tween the kinetic parameters for the glucuronidation of OH-PCBs a nd several physical parameters for these substrates (Table 4-5). The data for 4OHBP was not used si nce this compound is not a OH-PCB. The affinity of intestinal UGTs was negativ ely correlated with the Connolly solventaccessible surface area, the molecular surface area, solvent-excluded volume, ovality, dihedral angle, log P, and positively correlated with p Ka. The maximum rate of hepatic glucuronidation was negatively correlated w ith the Connolly solven t-accessible surface area, the molecular surface area, solvent-ex cluded volume, ovality, and log P, and positively correlated with p Ka (which showed a similar relationship with intestinal Vmax). Ovality was also significantly negatively correl ated with the maximum rate of intestinal glucuronidation of the OH-P CBs studied (Figure 4-5). A paired t-test performed in order to investigate the physicoc hemical parameters involved in the signif icant decrease in Vmax observed for the gl ucuronidation of OHPCBs with two chlorine atoms flanking the phenolic group revealed that, for OH-PCBs with this structural arrangement, p Ka was decreased ( p =0.02), while log P, and parameters indicating molecular size (CAA, CM A, CSEV, ovality) were all increased (all p <0.0001).

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52 Table 4-5. Results of regression analysis performed in order to investigate the relationship between the glucuronidat ion of OH-PCBs by catfish proximal intestine and liver and various estimated physical parameters. Physical Intestine Liver Parameter Statistic Vmax (app) Km (app) Vmax (app) Km (app) CAA R2 0.060 0.439 (-) 0.253 (-) 0.004 p -value 0.079 <0.0001 0.0005 0.685 CMA R2 0.056 0.423 (-) 0.249 (-) 0.002 p -value 0.087 <0.0001 0.0006 0.780 CSEV R2 0.047 0.396 (-) 0.236 (-) <0.001 p -value 0.118 <0.0001 0.0008 0.962 Ovality R2 0.114 (-) 0.431 (-) 0.286 (-) 0.068 p -value 0.014 <0.0001 0.0002 0.088 Dihedral R2 0.002 0.248 (-) 0.077 0.026 angle p -value 0.755 0.0002 0.068 0.296 log P R2 0.058 0.111 (-) 0.250 (-) 0.061 p -value 0.086 0.044 0.003 0.137 log D R2 0.011 0.035 0.015 <0.001 ( p H 7.0) p -value 0.467 0.271 0.490 0.963 p Ka R2 0.143 (+) 0.108 (+) 0.306 (+) 0.093 p -value 0.006 0.047 0.0007 0.063 Sign in parentheses indicates type of correlation where it achieved significance.

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53 1.360 1.385 1.410 1.435 0 1000 2000 3000 4000Vmax (int) Vmax (liv) ovalityVmax (pmol/min/mg protein) Figure 4-5. Relationship between Vmax for OH-PCB glucuronidation in intestine and liver and ovality Discussion In comparison to catfish intestine, catfish liver displayed higher rates of glucuronidation of OH-PCBs, however both organs collectively biotransform the OHPCBs studied with similar efficiency. This occurred because while the glucuronidation Vmax in the intestine was lower than in the liver, the affinity of intestinal UGTs for the OH-PCBs was higher than liver UGTs. However, the efficien cy of glucuronidation of 4 OHCB69 was seven times higher in the proxim al intestine; when the data for this substrate was excluded, the efficiency of glucuronidation was significantly higher ( p =0.01) in liver. The total UGT capacity in the liver is much greater than in intestine when the total content of microsomal protein in these two orga ns is taken into consideration. In fact, the levels of microsomal protein from liver were always higher than in the intestine of each individual fish studied, possibly because of the decreased amount of endoplasmic

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54 reticulum in enterocytes relativ e to hepatocytes (DePierre et al., 1987). Thus, the intestine appears to compensate for the lower glucur onidation capacity by expressing UGTs with a higher affinity. No relationship was established between Kms for the glucuronidation of OH-PCBs in liver and intestine. When individual OH-P CBs were considered, th ere were significant differences in efficiency. These results suggest that these two organs have different UGT isoform profiles, with the intestine possessing one or more isoforms that display greater specificity for OH-PCBs. Possible UGT isofor ms responsible may be catfish enzymes analogous to rat UGT1A1, UGT1A6 and UGT2B1 (Daidoji et al., 2005), and to plaice hepatic UGT1B1, which has been shown to c onjugate planar phenols (Clarke et al., 1992). The substrates 4 -OHCB69 and 4-OHCB39 were gluc uronidated with the highest efficiency in the intestine and liver respectively. 4 -OHCB35 showed the highest rates of glucuronidation in both liver and intestine. The poorest substrates were 4-OHCB14 in the intestine and 4 -OHCB112 in the liver. In contrast , rat liver glucuronidates 4-OHCB14 with the highest efficiency, relative to other OH-PCBs studied (Tampal et al., 2002). Overall, the efficiency of glucuronidation of the OH-PCBs by rat liver is higher than in catfish liver. While these dissimilarities may be ascribed to differences in UGT isoform type and expression due to the different specie s and tissues in the two studies, it may also indicate an increased susceptibility of catfish to the toxic effects of OH-PCBs due to an increased bioavailability. Compared to the OH-PCBs, 4-OHBP was th e poorest substrate for glucuronidation. This compound had the lowest Vmax in both liver and proximal intestine. The affinity for

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55 4-OHBP in the intestine was also the lowest. In the liver however, the Km was comparable to other OH-PCBs. These results ar e surprising in view of the fact that 4OHBP has been shown to be a good substrat e for glucuronidation using rat, guinea pig, beagle dog and rhesus monkey liver microsom es (Yoshimura et al., 1992), and human expressed UGTs (King et al., 2000; Ethell et al., 2002). In isolated rat hepatocytes, 4OHBP is a cytotoxic major metabolite of biphenyl, impairing oxidative phosphorylation (Nakagawa et al., 1993). These results suggest that this compound may be potentially more toxic to catfish than to mammals, unless cleared by another pathway such as sulfonation. While the decreased glucuronidation of 4-OHBP may be due to the lack of a specific phenol UGT isoform in catfish, the kno wn broad substrate sp ecificity of phenol UGTs, together with the obser ved higher rates of glucuroni dation for the OH-PCBs, leads us to hypothesize that this compound may be such a poor substrate due to its lower lipophilicity, as has been observed for othe r substituted phenols (Kim 1991). In fact, addition of a single chlorine atom fla nking the phenolic group (as represented by 4OHCB2) resulted in at least a tenfold increase in Vmax in both liver and intestine, with no significant change in Km (with respect to 4-OHBP). This increased lipophilicity (represented by an estimated log P increase from 3.2 to 3.8) appeared to impact the formation of the glucuronide and not the in itial binding of substrate to UGT. Good UGT substrates tend to be lipophilic compounds which are thought to diffuse through the endoplasmic reticular bilayer and reach the substrate-binding site in the lumenal N terminal part of the enzyme, which contai ns a region of strong interaction with the membrane (Radominska-Pandya et al., 2005) . For all the OH-PCBs studied, we only

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56 observed weak inverse correlations (R2<0.3) between log P and intestinal Km and liver Vmax. No significant relationship could be obser ved between parameters of lipophilicity and intestinal Vmax. The absence and weakness of such relationships may reflect the need for OH-PCBs with additional structural variati on to be included in studies of this type. Another explanation may be the perturbation of the lipid bilayer of the microsomes, resulting in rate-limiting partitio ning, which would not be present in vivo (Tampal et al., 2002). As the estimated p Ka of the OH-PCBs increased, so did hepatic and intestinal Vmax for glucuronidation. These results are in agreement with a previous OH-PCB glucuronidation study in rats (Tampal et al., 2002). Thus, a greater proportion of ionized OH-PCB molecules appear to have an adve rse effect on glucuronidation. Such charged molecules present at the active site of UGT may interfere with the charge-relay system that relies on a basic negatively charged resi due to deprotonate the phenolic group, prior to transfer of glucuroni c acid (Yin et al., 1994). Since the use of microsomal systems to elucidate structure-activity relationships involves incubations of substrate with a heterogeneous population of UGTs exhibiting different levels of expression and activity, it was not the inten tion of this study to attempt to predict the effect of molecular stru cture and physicochemical parameters on the glucuronidation of OH-PCBs, which is be tter achieved using individual isoforms. However, if any such effects can be observed at a microsomal level, th en it is likely that such processes are occurring in the organism, whose de toxification route depends on various UGTs metabolizing substrate simultaneous ly and not in isolation. This may help to further delineate the differe nt toxicokinetics of OH-PCBs.

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57 The p -OH-PCBs used in this study all had one or two chlorine atoms flanking the phenolic group. This structural motif is of interest since it imparts several toxic properties to these compounds. OH-PCBs w ith two flanking chlorines were found to be poorer substrates than compounds with one flanking chlorine atom, in both liver and intestine. Thus, for example, while 4 -OHCB35 was a very good subs trate for glucuronidation, addition of a second flanking chlorine (as in 4 -OHCB79) resulted in a greater decrease in Vmax than the addition of two adjacent chlorine substituents on the aphenolic ring (as in 4 -OHCB106). A comparison of the physicoc hemical parameters of the two different structural arrangements suggests that lipophilicity, p Ka, and molecular size may all be contributing to this effect on Vmax. The addition of a second chlorine atom imparts additional lipophilicity to the molecule and may increase positive charge on the phenolic carbon atom, which results in stronger binding to the active site (Wang 2005). This study did show a non-significant decrease in Km with the addition of the second chlorine atom for both organs. On the other hand, the 3,5-chlorine s ubstitution patte rn may interfere with the mechanism of glucuronidation because of steric hindrance, although this has been disputed (Mulder and Van Doorn 1975; Tampal et al., 2002). The estimated p Kas for OH-PCBs with two flanking chlorine substituents were significantly lower than similar molecules with one flanking chlorine atom. This is supported by limited experimental data s howing that OH-PCBs with two flanking chlorine atoms have p Ka values as low as 6.4 (f or 4´-OHCB39, Miller 1978). The population of OH-PCB molecules whic h are ionized at physiological p H is significantly more than OH-PCBs with one flanking chlorine atom, resulting in the adverse effect on

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58 the enzymatically-catalyzed charged relay sy stem described above. In studies conducted with rat liver microsomes, a decreased maximal rate of glucuronidation was also observed amongst OH-PCBs differing only in the number of chlorines flanking the phenolic group (1 pair of OH-PCBs in Tamp al et al., 2002; 2 pairs of OH-PCBs in Daidoji et al., 2005). According to Daido ji and co-workers (2005), UGT2B1 is the primary rat hepatic UGT isoform responsib le for metabolizing OH-PCBs with one flanking chlorine atom. UGT1A 1 appears to metabolize both, though with a preference for structures with two flanking chlorines These results are significant from a toxi cological standpoint si nce almost all the major OH-PCBs found in human plasma incorporate a 4`-hydroxy-3`,5`-dichloro structure (Sandau et al., 2002; Fangstrom et al., 2002; Hovander et al., 2002). It is possible that one reason for the persistence of these OH-PCBs may be a reduced rate of glucuronidation due to this structural arrangement. Two or more chlorine substituents that are ortho to the biphenyl bond cause the molecule to twist and assume a non-coplanar conformation. In the parent PCBs this leads to toxicological differences, such as loss of AhR agonist activity. The estimated dihedral angles for the compounds investigated in this study ranged from 36°-76°. The affinity of intestinal, but not hepatic, UGTs appeared to increase with the degree of twisting, suggesting that the predominant isoform(s) in catfish intestine binds more strongly to the more twisted OH-PCBs. While this may be additional evidence of differences with respect to isoform profiles between liver and intestine, the weakness of the relationship (R2~0.3) precludes using this result to solidly support th is hypothesis.

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59 Similar to what has been reported for the glucuronidation of OH-PCBs in rats (Tampal et al., 2002) and simple phenols by human UGT1A6 (Ethell et al., 2002), the maximal rate of hepatic glucuronidation decrea sed with increased steric bulk. In the case of intestinal glucuronidation this relations hip was weaker. The enzyme affinity of intestinal UGTs increased with increasing mo lecular size, perhaps because the bulkier molecules tended to be more lipophilic. Howeve r, in contrast, the affinity of the liver UGTs was not affected as much by the molecula r size, at least within the restricted size range offered by the OH-PCBs studi ed. At this point, no explanation for this discrepancy between these two tissues is forthcoming. Conclusions and Recommendations OH-PCBs are glucuronidated with similar efficiency by channel catfish liver and proximal intestine. There appear to be diffe rences in the UGT isozyme profile in both organs. The Vmax for both hepatic and intestinal gl ucuronidation was decreased with the addition of a second chlorine atom flanking the phenolic group, whic h is an arrangement typical of OH-PCBs that persist in organisms. Future research may be directed towards cloning, sequencing and characterizing these cat fish UGTs, in order to have a better understanding of the specificity of individua l UGT isoforms for particular chlorine substitution patterns in OH-PCBs.

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60 CHAPTER 5 CLONING OF UDP-GLUCURONOSYLTRANSFERASES FROM CHANNEL CATFISH LIVER AND INTESTINE Piscine UGT Gene Structure and Isoforms Fish are the most ancient vertebrate p hylum, and account for over 40% of all living vertebrate species (Clarke et al. 1992a). Clarke and co-workers (1992b) compared the hepatic glucuronidation of several xe nobiotics and endobiotics in plaice ( Pleuronectes platessa ) and rat ( Rattus norvegicus ), species that are separated by more than 350 million years of evolutionary diverg ence. Despite the fact that the plaice showed reduced glucuronidation activity toward s substrates such as morphi ne, bilirubin and steroids, weak immunological cross-reactivity was obt ained when anti-rat UGT antibodies were used, indicating the presence of conserved common structural motifs between the two vertebrates. Characterization of plaice UGT1B1 (Acce ssion number (AN): X74116), an isoform which conjugates planar phenols and is inducible by polyaromatic hydrocarbons (PAH), confirmed the strong degree of conservation in gross exon structure and amino acid character (signal peptide, membrane insert ion, and stop sequences) between fish and mammals. The greatest degree of similarity in amino acid sequence was found with UGT1 rather than UGT2 (Clarke et al., 1992b, Ge orge et al., 1998). Allelic variations in this UGT1B1 gene are presumed to be func tionally silent (George and Leaver 2002). While there is strong evidence for other dist inct isoforms conjuga ting bilirubin, estrogen and androgens, to date these have not been characterized. At least six distinct UGTs

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61 exhibited tissue-specific e xpression in plaice (Clarke et al., 1992c). UGT1B2 mRNA has recently been sequenced from marbled sole ( Pleuronectes yokohamae ) liver (AN: AB120133), and a partial sequence of an unide ntified UGT isoform has been obtained from the orange-spotted grouper ( Epinephelus coioides ) (AN: AY735003). The existence of a number of partial length sequence s of UGT homologues from zebrafish ( Dario rerio ) EST projects in GenBank provide evidence for the cDNA of 10 distinct UGTs. The absence of cDNAs with the same 3´seque nce and dissimilar 5´exon 1 coding sequence suggests the absence of alternative splicing of UGT1A genes as seen in mammals. Thus, George and Taylor (2002) ha ve suggested the existence of three family 1-related UGTs and another two related to the UGT 2 family in the zebrafish. In general, however, it appears that fish possess multiple UGTs with similar functional and structural properties to mammalian UGT. Toxicologically, it is important to know whether xenobiotic pollutants such as PAHs compete with steroids or bilirubin for the same active site on UGT, resulting in physiological perturbations in reproductive and/or liver func tion. For example, Atlantic salmon ( Salmo salar ) suffering from a multiple pollutant -induced jaundice were shown to have decreased bilirubin UGT activity (Georg e et al. 1992). Channel catfish are also exposed to pollutants (such as PAHs and PCBs) which accumulate in sediments. Thus, this organism may be a useful indicator of th e bioavailability of th ese pollutants in such sedimentary environments. In addition, the use of this fish in aquaculture makes it essential to understand every aspect of its detoxification mechanis ms, since these will ultimately impact human health.

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62 While no UGTs have yet been cloned and characterized from channel catfish, this species shows glucuronidation activity towards a variety of toxic xenobiotics, including monoand di-hydroxy metabolites of benzo[ a]pyrene and OH-PCBs (James et al., 2001; van den Hurk and James 2001; Gaworecki et al., 2004). As with othe r aquatic species, pollutants which are direct substrates for glucuronidation, such as pentachlorophenol, several OH-PCBs, 4-OH-heptachlorostyrene, and which have been shown to be estrogenic and thyroidogenic, have been dete cted in channel catfish (Li et al., 2003). Kinetic differences have been observed betw een hepatic and intestinal UGT activities, suggesting expression of diffe rent isozymes in these two organs. Thus, knowing more about the identity and substrate specificity of catfish UGTs will a ssist our understanding of the effect of glucuronidation on the contri butions of such metabolites to toxicity. Since the absence of cDNAs with the same 3´sequence and dissimilar 5´exon 1 coding sequence in fish suggests the absence of alte rnative splicing of UGT1A genes as seen in mammals (Gong et al., 2001), additional informa tion on piscine UGT gene structure is also important from a phylogenetic perspective. Hypothesis Multiple UGT isoforms are present in channel catfish liver and intestine Methodology (part 1) For convenience, a flowchart summarizing the various steps i nvolved in the cloning process is shown in Figure 5-1. Because the study utilizing the gene specific primers was dependent on an initial study which utilized de generate primers and le d to the cloning of partial sequences of UGT, the methodology and results sections are split correspondingly in two parts.

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63 UGT cDNA pGEM T-Easy vector+recombinant plasmidE.coli JM109bacterial/plasmid replicationUGT clonesplasmid purificationcatfish liver/intestine1. RNA isolation 2. RT-PCR, using degenerate prim ers based on consensus sequencesSequencing of partial length UGTs BLAST searchtransformation ligation 1. Design GS primers 2. 5` and 3` RLM-RACE 3. Clone and sequenceSequence overlap1. Design GS primers 2. PCR with Super Taq Plus 3. Clone and sequenceFull-length cDNA clone Figure 5-1. Summary of methods used to clone channel catfish UGT Animals. A single female adult catfish was s acrificed. Total weights of liver and intestinal mucosa were recorded. Tissues were immediately proce ssed for RNA isolation. RNA isolation. Approximately 0.1g of tissue from the liver and proximal intestinal mucosa were homogenized in separate tubes with 1 mL Trizol® reagent and placed on ice. The homogenates were incubated for 5 mi n at room temperature (15-30°C) to enable complete dissociation of nucleoprotein comp lexes. Chloroform, 0.2 mL, was added and

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64 the tubes were shaken vigorously by hand fo r 15 seconds and then incubated at room temperature for 2-3 min. The samples were then centrifuged at 12,000g for 15 min at 28°C. This separated the solution into an aqueous phase containing the RNA and an organic phase containing DNA. The colorless upper aqueous phase wa s transferred to an RNase-free tube. The RNA was precipitated by the addition of 0.5 mL propan-2-ol. The samples were then incubated at room temp erature for 10 min, followed by centrifugation at 12,000g for 10 min at 2-8°C. The RNA precip itate was now visible as a gel-like pellet on the side and bottom of the tube. The supernatant was removed and the RNA pellet was washed once with 1 mL 75% ethanol. The sa mple was vortex-mixed and centrifuged at 7,500g for 5 minutes at 2-8°C. The RNA pellet was left to air-dry for a few minutes following decantation of the ethanol. The RNA was dissolved in 100 L RNase-free water for intestine, and 200 L RNase-free water for liver (since the solution in this case appeared to be more concentrated), by pa ssing the solution a few times through a pipette tip. The solution was then incubated for 10 mi nutes at 55-60°C. The samples were stored at -80°C. The purity of the RNA was checked by running the sample on 1% agarose gel (with 9.5% formaldehyde) and 10x MOPS buffer . Bands corresponding to the 28S and 18S ribosomal subunits were observed. The purity of the RNA was also checked by diluting the sample in 10mM Tris HCl, p H 7.5 and measuring the A260/A280 absorbance ratio (ideally should be between 1.8 and 2.1). DNase treatment of RNA samples. This procedure was done in order to remove contaminating DNA from RNA preparations, a nd to subsequently remove the DNase and divalent cations from the sample. Portions of the RNA solutions were diluted to 100 g/mL with RNase-free water. The Ambion ® (Austin, TX) DNA-removal kit was used.

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65 The reaction mix, consisting of 25 L RNA, 2.5 L 10xDNaseI buffer, and 3 L DNase I was incubated at 37°C for 1 hour. DNase inactivati on reagent, 5 L, was added by means of a wide pipette tip (due to the thick consis tency of this reagent). The tubes were then incubated for 2 min at room temperature, with gentle flicking. The tubes were then centrifuged at 10,000g for ~1 min to pellet the DNase inactivat ion reagent. The supernatant containing the RNA was transferred to a new RNase-free tube and stored at 80°C. Generation of cDNA library. The Retroscript® reagent kit manufactured by Eppendorf (Westbury, NY) was empl oyed in order to heat-denat ure the RNA. To each of the two tubes were added 10 L liver or intestinal RNA (equivalent to 1 g) and 2 L random decamers. The tubes were mixed, centr ifuged briefly and heated for 3 min at 7085°C in the thermocycler. Tubes were removed and left on ice for 1 minute. They were centrifuged and put on ice again. The following components were added to each tube: 2 L 10xRT buffer, 1 L dNTP mix (10mM), 0.5 L RNase inhibitor, 1 L reverse transcriptase, and RNase-free water to 20 L. The tubes were gently mixed and centrifuged briefly. They were placed in the thermocycler for 1 hr at 42-44°C, followed by 92°C for 10 min. The resulting cDNA was either stored at -20°C or subjected to a second round of PCR (liver, see below; for th e intestine this procedure was performed a few days after cDNA generation). Degenerate primer design. A characteristic ‘signature sequence’, 44-amino acids long, probably corresponding to the UDPGA bi nding site, has been shown to be highly conserved amongst mammals and other vertebra tes (Mackenzie et al., 1997). The relevant amino acid and nucleotide sequences were compar ed in 4 species of fish using ClustalW.

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66 The species investigated were Pleuronectes platessa UGT1B1, P.platessa UGT, Pleuronectes yokohamae UGT1B2, Epinephelus coiodes UGT and Danio rerio UGT. Five primers were designed which could hyp othetically bind to this sequence. The application of exclusion crite ria (degeneracy <100-fold, poor or no matches with fish sequences resulting from BLASTn searches , %GC content <40%, potential to selfdimerize < -20 kcal/mol) resulted in the sele ction of two primers, designated as UGT_R3 and UGT_R4, and chosen to be reverse primers (Table 5-1). An additional reverse primer (UGT_R5) was chosen due to its low degenera cy (4-fold) and its complementarity to the highly conserved N-terminal domain downs tream of the signature sequence. Five additional primers (UGT_F3-7) were also chosen based on these same criteria. Since these primers were complementary to sequences upstream of the signature sequence, they were selected to be forw ard primers (Table 5-1). Table 5-1. 5' 3' Sequences of degenerate primers chosen. ID Sequence Direction UGT_F3 GTGGTSCTGGTSCCYGAAASYAGY Forward UGT_F4 CTTACWGAYCCMTTCYTKCCSTGYGGC Forward UGT_F5 AACATGGTYYWWATYGGRGGYATCAACTGT Forward UGT_F6 ATYGGRGGYATCAACTGTGCA Forward UGT_F7 GAGTTTGTSVAHGGCTCWGGA Forward UGT_R3 AAACAGHGGRAACATCAVCAT Reverse UGT_R4 YCCYTGSTCKSCAAACAGHGG Reverse UGT_R5 GTGRTACTGRATCCAGTTCAG Reverse ___________________________________________________________________

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67 The primer pairs were selected in such a way that their melting temperatures did not vary by more than 6°C and their pote ntial to heterodimerize was more than –20 kcal/mol (Table 5-2) Table 5-2. Primer pairs chosen, showing a nnealing temperature and estimated amplicon length Pair Forward Reverse Amplicon length (bp)1 T (°C) 3 UGT_F6 UGT_R5 618 52.6 4 UGT_F7 UGT_R3 288 53.8 5 UGT_F6 UGT_R3 339 53.8 6 UGT_F7 UGT_R5 567 52.6 7 UGT_F5 UGT_R4 363 61.1 8 UGT_F3 UGT_R4 1003 61.1 9 UGT_F4 UGT_R4 729 61.1 __________________________________________________________ 1 Based on Danio rerio UGT sequence (Accession number NP_998587.1) PCR amplification of UGT cDNA. A 10 M solution of each primer in nucleasefree water was made up. Each PCR tube consisted of 2 L DNA template (from catfish), 2 L forward primer, 2 L reverse primer, 0.5 L Taq DNA polymerase (5U/ L, in Mg 10x buffer), 1 L dNTP mix (10mM), 5 L 10xPCR buffer, and nuclease-free water up to 50 L. Prior to the initiation of the PCR reaction, with the tubes in place, the thermocycler lid was heated for two minutes at 110°C to prevent sample evaporation. Thermocycler parameters (utilizing a gradie nt PCR program to adjust for the different optimal annealing temperatures required by th e various primer pairs) were as follows:

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68 Stage Temp /°C Duration/min Initial Denaturation 94 2 Denaturation 94 0.5 Annealing 57±5 (L); 55±5 (I)1 0.5 Extension 72 1.0 Final extension 72 7.0 _____________________________________________________ 1 annealing temperatures used fo r: L, liver; I, intestinal cDNA The program consisted of 35 cycles of denaturation, annealing and extension. The PCR products were subjec ted to electrophoresis on 1% agarose gel at 100V (in 1x TAE buffer (40 mM Tris base, 5 mM sodium acetate, 1 mM EDTA, p H 8.0)) using 30 L of PCR product; a 100bp DNA ladder was us ed for size estimates. The DNA bands were visualized by placing on a UV transi lluminator and recorded by photography. Recovery of PCR product fr om gel and purification. The desired DNA band was excised from the gel using a clean scal pel and transferred to a pre-weighed 1.5mL microcentrifuge tube. The Wizard ® SV Gel Clean Up system (Promega, Madison, WI) was used to purify the PCR product by centrifug ation. Membrane binding solution (4.5 M guanidine isothiocyanate, 0.5 M potassium acetate, p H 5.0), 10 L per 10 mg gel, was added to the gel slice. The mixture was vortexed and incu bated at 51°C for 10 min in order to dissolve the ge l slice. The tube was then briefl y centrifuged at ro om temperature. For every solution (derived fr om the cut gel slices), the following procedure was adopted. One SV Minicolumn was placed in a coll ection tube. The dissolved gel mixture was transferred to the SV Minicolumn assembly a nd incubated for 1 min at room temperature. The assembly was centrifuged in a microcentrifuge at 16,000 g for 1 minute and the liquid

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69 in the collection tube was discarded. The column was washed by 700 L of Membrane Wash Solution (10 mM potassium acetate, p H 5.0, 16.7 M EDTA, p H 8.0, 80% ethanol). The assembly was centrifuged for 1 min at 16,000g, and the collection tube was emptied. Another 500 L Membrane Wash Solution was added to the assembly, followed by centrifugation for 5 minutes at 16,000g. Th e collection tube was emptied, and the collection tube was recentrifuged for 1 minut e to dry the column. The SV Minicolumn was transferred to a clean 1.5 mL microcentrifuge tube and 50 L nuclease-free water was applied to the column and incubated for 1 minute at room temperature. The Minicolumn/micro-centrifuge tube wa s centrifuged for 1 minute at 16,000g. The Minicolumn was discarded and the tube contai ning the eluted DNA wa s stored at -20°C. A portion of this DNA was diluted w ith 10 mM Tris-HCl, 1 mM EDTA, p H 8.0, and used to calculate the DNA concentra tion by its absorbance at 260nm. Ligation and transformation of E.coli . LB plates with ampicillin were first prepared. LB Agar, 8.75 g, was weighed and dissolved in 250 mL, and the p H was adjusted to 7.2 with NaOH. The solution was autoclaved for 30 min at 120°C. After the medium cooled to around 50°C, ampicillin was added to a final concentration of 100 g/mL. Some of the medium, 30-35 mL, was poured into 85-mm Petri dishes and the agar left to harden. The plates were left ove rnight at room temperature and subsequently stored in an inve rted position at 4°C The ligation was performed using the p-GE M T-Easy Vector System® supplied by Promega. The volume of PCR product to be us ed in the ligation reaction could not exceed 3 L. The amount required was calculated from the following equation, which assumes that the optimal insert:vector molar ratio is 3:1:

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70 50 ng vector x a kb insert x 3 = ng insert required 3.0kb vector 1 where a is the approximate size of amplified insert Because of this limit in sample volume, th e amount of insert actually used was less than that recommended since the concentra tion of purified DNA was relatively low. The ligation reactions were setup as follows (all volumes in L) in 0.5 mL tubes: Standard Positive Background Component Reaction Control Control 2x Rapid Ligation Buffer, T4DNA ligase 5 5 5 pGEM T-Easy Vector (50 ng) 1 1 1 PCR-product (9 ng) 3 ----Control insert DNA --2 --T4 DNA ligase (3 Weiss units/ L) 1 1 1 DNase-free water 0 1 3 The ligation buffer was mixed vigorously be fore use. The reactions were mixed by pipetting and incubated for 1 hour at room te mperature, followed by storing overnight at 4°C. JM109 high-efficiency competent cells ( 1x108 cfu/ g DNA; Promega) were used for transformation. The following procedure was performed using aseptic technique (sterile tips and tubes, use of Bunsen fl ame to create upward convection in work area). The tubes containing the ligation reactions were centrifuged for 1 minute at 10,000 rpm and placed on ice. Another t ube (transformation control, TC) was set up on ice; this contained 0.1 ng uncut plasmid (0.1 L of 0.1 mg/ L solution used) in order to determine the transformation efficiency of the competen t cells. Tubes containing frozen aliquots of JM109 cells were removed from -80°C storage and placed on ice until thawed (~5 min).

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71 The cells were mixed by gentle flicking of the tubes. Each ligation reaction, 2 L, was added to a 1.5 mL microcentrif uge tube on ice, followed by 50 L of cells (100 L were added to the TC). The tubes were mixed by ge ntle flicking and left on ice for 20 minutes. The cells were heat-shocked by placing in a 42°C water bath for 45-50 sec. The tubes were then returned to ice for 2 minutes. S. O.C medium (Invitrogen Corp., Carslbad, CA), 950 L, was added to each tube (900 L was added to the TC). The tubes were then incubated for 1.5 h at 37°C with shaking (~ 150 rpm). The ampicillin/LB plates were removed from 4°C storage, 100 L of 100 mM isopropylthiogalactoside (IPTG, a galactosidase inducer) and 20 L of 50 mg/mL 5-bromo-4-chloro-3-indolylD galactoside (X-Gal, hydrolyzed by -galactosidase to yield a bl ue product) were added, and the mixture spread on each plate. The ag ar was allowed to absorb these compounds for 30 min at 37°C. Samples, 100 L, of each transformation cu lture were transferred to, and streaked on, duplicate LB/ampicillin /IPTG/ X-Gal plates; for the TC, 20 L of tube culture was diluted with 180 L of S.O.C. medium, and 100 L of this dilution was applied to the agar plates. The plates were incubated overnight (~16h) at 37°C. Plates were then stored at 4°C for 30 minutes to facilitate color development. The white colonies should contain plasmids with the in sert, while the blue colonies do not contain the insert since the protein-encoding sequence of the lac Z gene in the vector is not interrupted by the insert and hence can lead to -galactosidase synthesis and catalysis of the X-Gal reaction. Colony PCR and culturing E.coli with insert of interest. Two white and one blue colony from each plate were picked by a sterile wooden toothpick, which was inserted in a PCR tube containing 5 L 10x PCR buffer, 5 L 10mM dNTP mix, 1 L

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72 PUC/M13 forward primer, 1 L PUC/M13 reverse primer, 0.5 L Taq DNA polymerase, and DNase-free water to 50 L. The pGEM T-Easy Vector contains binding sites for the PUC/M13 primers. Thermocycler parameters were as shown previously, but with an annealing temperature of 55°C (no temperatur e gradient). PCR products were run on 1% agarose gel at 100V, using 15 L of the PCR product. Samples from colonies which showed th e presence of insert on the gel were extracted by an ethanol-flame sterilized me tal hoop and dispensed into 14 mL sterile, round-bottomed Falcon tubes containing 4 mL of LB medium with ampicillin by swirling. The tubes were incubated with shaking for 16-20 h at 37°C. Purification of plasmid DNA. A sample, 850 L, of each culture medium was diluted up to 1000 L with sterile glycerol and stored at -80°C. The rest of the culture medium was dispensed in 1.5 mL microcentr ifuge tubes, which were centrifuged for 2 min at 10,000g. The supernatant was poured off and the tubes were blotted upside-down on a paper towel to remove excess media. For plasmid purification, the Promega Wizard Plus Minipreps ® DNA Purification System wa s used. The cell pellets were resuspended in 200 L of cell resuspension solution (50 mM Tris-HCl ( p H 7.5), 10 mM EDTA, 100 g/mL RNase A). Cell lysis (0.2 M NaOH, 1% SDS) solution, 200 L, was added and the tubes inverted 4 times to clarify th e solution. Neutralization solution (1.32 M potassium acetate, p H 4.8), 200 L, was added and mixed by inverting the tubes for 4 times, resulting in a white precipitate. Th e lysate was centrifuged at 10,000g for 5-20 minutes, depending on whether a ce ll pellet was clearly visible. One Wizard® Minicolumn was prepared for every Miniprep. A plunger was removed from a 3 mL disposable syringe and set aside. The syringe barrel was attached

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73 to the Luer-Lok® extension of the Minico lumn. DNA purification resin (7 M guanidine HCl), 1 mL, was pipetted in the barrel, follo wed by the cell lysate. The syringe plunger was inserted in the barrel and used to push the slurry through the Minicolumn. The syringe was detached from the Minicolumn and the plunger removed from the syringe barrel. The barrel was then reattached to th e Minicolumn. Column wash solution (80 mM potassium acetate, 8.3 mM Tris-HCl ( p H 7.5), 40 M EDTA, 55% ethanol), 2 mL, were pipetted into the barrel of the Minicolumn/s yringe assembly, and the solution was pushed through the Minicolumn by the plunger. The syringe was removed, and the Minicolumn was transferred to a 1.5 mL microcentrifuge tube, which was centrifuged at 10,000g for 2 min to dry the resin. The Minicolumn was tr ansferred to a new 1.5 mL microcentrifuge tube and 50 L nuclease-free water was added to the column and left for 1 minute. The DNA was eluted by centrifuging at 10,000g for 20 sec. The Minicolumn was removed and discarded, and the DNA solution stored at -20°C. Products were visualized by 1% agarose gel electrophoresis run at 100V, using 3 L of purified DNA and 10 L quantitative 1kb plus DNA ladder (0.5 g). Digestion with ecoRI . To ensure that the two DNA bands seen in the purified plasmid DNA run on agarose gel were due to supercoiling of the DNA and not contamination, the plasmid DNA was digested with the restriction enzyme ecoRI (pGEM T-Easy Vector has restriction sites on e ither side of the insert). Plasmid DNA, 3 L, was added to a tube containing 0.2 L acetylated BSA, 2 L 10x buffer, and 14.3 L DNasefree water, and mixed by pipetting. ecoRI restriction enzyme (12 U/ L), 0.5 L, was added and the solution mixed by pipetti ng. The tubes were briefly centrifuged and

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74 incubated for 2h at 37°C. The products were run in 1% agarose at 100V, using all the incubation mixture. DNA sequencing and data processing. The concentration of the purified plasmid DNA was determined prior to submission for sequencing. The DNA sequencing core requires 1.5 g DNA for adequate processing. Cl oned DNA sequences obtained were then compared with nucleotide sequences in GenBank using the BLASTn tool provided online ( http://www.ncbi.nlm.nih.gov/BLAST ). Multiple sequence comparisons were done with SeqWeb, while two-sequence compar isons were done with the BLASTn 2.2.12 program. Results and discussion (part 1) Primer pairs 4 and 6 successfully amplified cDNA from catfish liver; while primer pair 4 amplified cDNA from proximal intestine. The size of the amplicons were approximately 300bp (pair 4) and 600bp (pair 6) in size, with the gel-clean up system effectively removing primer dimers and othe r contamination (Figur e 5-2). The controls indicated that ligati on and transformation of the plasmid into E.coli were successful. Purified plasmid DNA was obtained from se veral colonies (Figure 5-3), which were denoted as L1-L8 for the liver and I1-I4 for th e proximal intestine. Th e two bands seen in these gels, did not represent contamination, as verified by the restriction digest of the plasmid, which resulted in a band correspondi ng to the vector and one corresponding to the smaller insert (Figure 5-4).

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75 A B 3 2 1 ladder ladder 3 2 1 ladder Figure 5-2. Products of PCR reaction. 1(from intestine), 2 and 3 (from liver) A. pre-cleanup; B. post-cleanup with the gel clean-up system. 100kb ladder shown for size estimation. 500

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76 Liver L8 L7 L6 L5 L4 L3 L2 L1 1kb ladder Intestine I4 I3 I2 I1 1kb ladder Figure 5-3. Plasmid DNA obtained from cultu res transformed with vector containing inserts from liver and intestine. 2,500 2,500

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77 100bp ladder L8 L7 L6 L5 L4 L3 L2 L1 1kb ladder Figure 5-4. Product of ecoRI dige st of purified plasmids c ontaining liver inserts L1-L8 The DNA sequences obtained are detailed in Appendix A. The results of the BLASTn search (best five sequences for each insert) are summarized below (Table 5-3). Very good matches were obtained with the Tetraodon nigroviridis cDNA, as well as Pleuronectes yokohamae UGT1B2, several Danio rerio sequences and Strongylocentrotus purpuratus (sea-urchin) UGT2B sequences. There was also a good similarity between the longer insert and the mammalian UGT1A sequences. Better matches were obtained with the l onger cDNA insert obtained from the liver (95 sequences with score >50) than with the shorter insert from liver or intestine (9 sequences with score >50). 3,000 300 600

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78 Table 5-3. Results of BLASTn search of cloned putative partial UGT sequences Accession no. Short description Insert Score (bits) E-value CNS0EY06 Tetraodon nigroviridis , full length cDNA L1 123 3E-25 L4 115 8E-23 L7 113 6E-22 I1 107 2E-20 CNSOEVYF Tetraodon nigroviridis , full length cDNA L1 123 3E-25 L4 115 8E-23 L7 113 6E-22 I1 107 2E-20 AB120133.1 Pleuronectes yokohamae UGT1B2 mRNA L7 85.7 1E-13 L1 67.9 2E-08 L4 67.9 2E-08 I1 60.0 4E-06 AF104339 Macaca fascicularis UGT1A01, mRNA L7 63.9 5E-07 BC109404.1 Danio rerio cDNA clone L7 61.9 2E-06 BC100055.1 Danio rerio cDNA clone L1 60.0 4E-06 I1 52.0 1E-03 BX005348.9 Danio rerio DNA sequence from clone L1 60.0 4E-06 I1 52.0 1E-03 XM 792456.1 Strongylocentrotus purpuratus , UGT2B34 L4 54.0 3E-04 XM792428.1 Strongylocentrotus purpuratus , UGT2B17 L4 54.0 3E-04 SeqWeb analysis of the sequences showed that the short inserts were almost identical with almost all the differences bein g located in the primer regions and thus may be attributed to the degenerate nature of the primers. Sequence L1 was found to be 98% similar to both sequences L7 and I1. While this implied that all these sequences are derived from the same isozyme, this could not be ascertained since most of the sequence differences between UGT isoforms arise from the N-terminal (substrate-binding) domain and only that part of the gene which codes for the highl y conserved C-terminal domain was cloned.

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79 Methodology (part 2) The next step in the cloning study was thus to design GSPs in order to extend the partial UGT sequences obtained so far to the full-length gene. Overview of RLM-RACE RNA-ligase mediated rapid amplificati on of cDNA ends, or RLM-RACE is a procedure used to extend a known DNA sequence towards its 5 and its 3 ends (Maruyama and Sugamo, 1994; Shaefer 1995). In 5 -RACE, total RNA is treated with calf intestinal phosphatase (CIP) to remove free 5'-phosphates from molecules such as ribosomal RNA, frag mented mRNA, tRNA, and contaminating genomic DNA. The cap stru cture found on intact 5'-ends of mRNA is not affected by CIP. The RNA is then trea ted with tobacco acid pyrophosphatase (TAP) to remove the cap structure from full-le ngth mRNA, leaving a 5'-monophosphate. A 45 base RNA Adapter oligonucleotide is ligat ed to the RNA popula tion using T4 RNA ligase. The adapter cannot ligate to dephosphor ylated RNA because these molecules lack the 5'-phosphate necessary for ligation. During the ligation reaction, th e majority of the full-length decapped mRNA acquires the adapter sequence as its 5'-end. A randomprimed reverse transcription reaction and ne sted PCR then amplifies the 5'-end of a specific transcript (Figure 55). The Ambion kit used in th is study provided two nested primers corresponding to the 5'-RACE Adapte r sequence, while two nested antisense primers were designed to be specific to the target gene. In 3 -RACE, first-strand cDNA is synthesi zed from total RNA using the supplied 3'-RACE Adapter. The cDNA is then subj ected to PCR using one of the 3'-RACE primers which are complimentary to the anchored adapter, and a user-supplied primer for the gene under study (Figure 5-5). Although 3'-RACE may not require a nested PCR

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80 reaction, this may also be performed if no si gnificant amplicons ar e detected after the outer PCR. 5`-PO4-AAAAA G--P--P--P-CIP AAAAA G--P--P--P-TAP AAAAA 5`-RACE adapter AAAAA 5`-RACE adapter 5`-RACE adapter CIP treatment to remove 5`PO4from degraded mRNA, rRNA, tRNA, and DNA TAP treatment to remove cap from full-length mRNA 5` RACE Adapter Ligation to decapped mRNA reverse transcription PCR 5`RLM-RACE 3` RACE AAAAA G--P--P--P-reverse transcription with 3` RACE Adapter AAAAA G--P--P--P-NVTTTTT-adapter PCR G--P--P--P-NVTTTTT-adapter Figure 5-5. 5 RLM-RACE and 3 RACE

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81 Design of gene-specific pr imers (GSPs) for initial 5 -RACE study. The initial primers used for RACE were designed to be 20-24 bases in le ngth, with 50% G:C content, and with no secondary structure. Prim ers contained less than 3G or C residues in the 3`-most 5 bases, and did not have a terminal G at the 3`-end. An online oligonucleotide analyzer ( www.idtdna.com ) was used to determine whether potential primers self-hybridized or hybridized to th e primers supplied with the RLM-RACE Kit. Figure 5-6 shows where the gene-specific prim ers and the primers supplied with the kit should be positioned with respect to the DNA template. ~150 bp 5' RACE Adapter 5`RACE outer primer 5`RACE inner primer UGT-specific 5`primer 5`RACE UGTspecific inner primer 5`RACE UGTspecific outer primer 5` RACE 3` RACE 3`RACE outer primer 3`RACE inner primer 3' RACE Adapter 3`RACE UGTspecific outer primer 3`RACE UGTspecific inner primer Figure 5-6. Primer positions for 5 and 3 -RACE.

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82 The DNA templates selected were those identified from the previous study, that is, the sequence isolated from liver (L6) and inte stine (I4). The primers used in this initial study are shown in Table 5-4. Table 5-4. Gene-specific primers us ed in initial 5`RLM-RACE study. GSP ID Sequence (5 3 ) Start position1 PCR step GSP_OUT TGCTCTGAGGTCAGGTCGAA 397 Outer GSP_INN ACAGATACCCTCGTAGATGCCA 280 Inner 1 From 5 end of sense strand of partial sequence L6 Based on homology with the complete sequences of Pleuronectes platessa UGT1 (PPL249081) and Macaca fascicularis UGT1A1 (AF104339) it was estimated that, for the UGT sequence isolated from catfish liver, this sequence needed to be extend by ~920 bp to the 5`-end, and ~183 bp to the 3`-end. Unfortunately, the use of these primers led to sequences which still lacked the 5 -end (L15R, L25R, L35R, I15R, I25R, I35R; see Appendix A). In addition, a high degr ee of non-specific binding was noted. Design of GSPs for succeeding 5 and 3 -RACE study. A new batch of GSPs was designed (Table 5-5) using different crite ria than the ones mentioned above in an attempt to improve sensitivity. Primer 3.0 Software ( http://frodo.wi.mit.edu/cgibin/ primer3/primer3_www.cgi ) was used to design primers, based on the following criteria: a. For 5 -RACE, GSPs with a GC-clamp at the 3 -end in order to reduce nonspecific binding were used, b. The outer and inner primer melting temp eratures for the GSPs were within a degree of the RACE kit supplied primers, c. For 5 -RACE, the inner primer was long (~27 bp) in order to reduce nonspecific binding, and

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83 d. The primers were designed to anneal close (50-75 bp) to the existing 5 -end to avoid large overlaps. Different sets of primers were designed based on the cDNAs obtained by the study involving the degenerate prim ers (I4) and the initial 5 -RACE study (I35R and L25R). Table 5-5. Gene-specific primers us ed in succeeding RLM-RACE study GSP ID Sequence (5 3 ) RACE Start1 PCR step (a) Liver L25R UGT_5OUT1 ATTGGGCATTACAGGTCTCG 5` 119 Outer UGT_5INN1 CGAGGACGTCTCTGAACGTAACATCC 5` 51 Inner UGT_3OUT1 GATTCCTCAGAGGGTTCTGT 3` 320 Outer UGT_3INN1 GGGGTCATTCCCAAAGACAT 3` 351 Inner (b) Intestine I4 UGT_5OUT2 GCCGTTACAGATA CCCTCGT 5` 282 Outer UGT_5OUT2A GTATCGCCACAGAACCCTCT 5` 138 Outer UGT_5INN2 ACACGAAGGAGCTCAAAGTGAACACG 5` 61 Inner UGT_5INN2A GCCACTTCATCAC TTTGACATTTTCAGG 5` 190 Inner UGT_3OUT2 AATGTCAAAGTGATGAAGTGG 3` 169 Outer UGT_3OUT2A GACATTCCTGAAAATGTCAAA 3` 157 Outer UGT_3INN2 CCCAAGGCTAAGGTGTTCATC 3` 217 Inner UGT_3INN2A GACCTCTTAGCACACCCCAAG 3` 202 Inner (b) Intestine I35R UGT_5OUT3 TGTTAATGACCTTCGGTGTGA 5` 236 Outer UGT_5OUT3A ATGACCTTCGGTGTGAGTTTT 5` 231 Outer UGT_5INN3 AAACCTAAGAGGTCATTCTGCGGAAGC 5` 70 Inner UGT_5INN3A ATGGGGACCGGGTGTCTATTTATTACG 5` 115 Inner UGT_3OUT3 TTTCCAGCTAACACTACTTGG 3` 178 Outer UGT_3OUT3A TTACACGTCCTCTAACCGTAA 3` 73 Outer UGT_3INN3 CCCAAGGCTAAGGTGTTCATC 3` 355 Inner UGT_3INN2A CCATGGCATCTACGAGGGTAT 3` 390 Inner 1 Start position from partial DNA sequences obtained so far

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84 5` RLM-RACE procedure Calf intestinal phosphatase (CIP) treatment. Total RNA (not DNase treated) (2 L for liver; 1 L for intestine), 10 g, as well as 10 g of control RNA (mouse thymus) were gently mixed with CIP buffer, CIP, and nuclease-free water in a total volume of 10 L. The mixture was incubated at 37°C for 1 hour, and terminated by the addition of 15 L ammonium acetate solution. A 115 L volume of nuclease-free water was added, followed by 150 L acid phenol-chloroform. The mixtur e was then vortexed thoroughly and centrifuged for 5 minutes at room temperature and at 10,000g. The aqueous phase was transferred to a new tube, 150 L chloroform were added, and the mixture was thoroughly vortexed and centr ifuged for 5 minutes at 10,000g. The top aqueous layer was transferred to a new tube, 150 L isopropanol were added, followed by thorough vortexing and chilling on ice for 10 minutes . The mixture was then centrifuged at maximum speed (16,000g) for 20 minutes. The pellet was rinsed with 0.5 mL cold 70% ethanol and centrifuged for 5 minutes at 16,000g . The ethanol was carefully removed and discarded, and the pellet was allowed to air dry (but not completely). The pellet was resuspended in 11 L nuclease-free water and placed on ice. At this point 1 L of the CIP-treated RNA was reserved for the “m inus-TAP” control reaction. This RNA was carried through adapter ligation, reverse transc ription and PCR in order to demonstrate that the products generated by RLM-RACE we re specific to the 5`-ends of decapped RNA. Tobacco Acid Pyrophosphatase (TAP) treatment. CIP’d RNA, 5 L, was gently mixed with TAP, 10XTAP buffer and nucleas e-free water in a total volume of 10 L. The mixture was incubated at 37°C for 1 hour.

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85 5`RACE Adapter Ligation. CIP/TAP-treated RNA, 2 L, and 2 L of CIP-treated RNA (minus-TAP control) was gently mixed with 1 L 5`RACE adapter (5`GCUGAUGGCGAUGAAUGAACACUGCGUU UGCUGGCUUUGAUGAAA 3`), 1 L 10XRNA Ligase buffer (before use, the buffer was quickly warmed by rolling it between gloved hands to resuspend a ny precipitate), T4 DNA Ligase (2.5 U/ L), and nuclease-free water in a total volume of 10 L. The mixture was incubated at 37°C for 1 hour, after which it was stored at -20°C. Reverse transcription (RT). Ligated RNA, 2 L, or minus-TAP control were gently mixed with 4 L dNTP mix, 2 L random decamers, 2 L 10XRT buffer, 1 L RNase inhibitor, 1 L M-MLV reverse transcriptase, a nd nuclease-free water in a total volume of 20 L. The mixture was incubated at 42°C (or 50°C, see results) for 1 hour. The reactions were stored at -20°C. Outer PCR. Each tube contained: 1 L RT reaction, 5 L 10XPCR buffer, 4 L dNTPmix (4 mM), 2 L gene-specific or outer c ontrol (reverse) primer (10 M), 2 L outer (forward) primer (10 M) (5`-GCTGATGGCGATGAATGAACACTG-3`), 0.25 L Taq DNA polymerase (5 U/ L), and nuclease-free water in a total volume of 50 L. A minus-template control was also included to ensure that one or more of the PCR reagents was not contaminated with DNA.

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86 Thermocycler parameters were as follows (Lid heating at 110°C): Step Stage Temp/°C Duration/min 1 Initial denaturation 94 3 2 Denaturation 94 0.5 3 Annealing 59 ± 21 0.5 4 Extension 722 13 5 Final extension 72 7 35 cycles of steps 2 – 4 were performed 1,2,3 These parameters were frequently changed to optimize the PCR. The values given above are representative of parameters us ed with the GSP_OUT and control primers. Inner nested PCR. A mixture was prepared, identical to the one for outer PCR, except that the DNA template was now 1 L of the outer PCR, and 2 L each of both inner primers. The sequence for the inner 5` RACE primer supplied with the kit was 5`CGCGGATCCGAACACTGCGTT TGCTGGCTTTGATG 3`. The thermocycler parameters were similar except for the annealing temperature, which was typically higher than the one used for the outer PCR. 3` RACE procedure Reverse transcription. The following components were assembled in a nucleasefree microfuge tube: 1 g total RNA (DNase-treated) from intestine or liver or control (mouse thymus RNA), 4 L dNTP mix, 2 L 3`RACE Adapter (5` GCGAGCACAGAATTAATACGACTCACTATAGG T12VN 3`), 2 L 10XRT buffer, 1 L RNase inhibitor, 1 L M-MLV reverse transcriptase, and nuclease-free water to 20 L. The reaction was mixed gently and incubated at 42°C or 50°C for 1 hour. PCR. The procedure for the outer and inner PCR was similar to the one performed for 5`-RACE, the only difference being the GSP and the kit-supplied primers used. The

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87 sequences for the latters were as follows: Outer 5`-GCGAGCACAGAATTAATACGA CT-3`, Inner 5`-CGCGGATCCGAATTAATACGACTCACTATAGG-3` PCR amplification of entire UGT gene Elucidation of the complete gene seque nce for liver UGT from catfish by RLMRACE (via partial sequence overlap) enabled the design of gene specific primers which are complementary to the gene itself as well as the untranslated region. The primers used are shown in Table 5-6. All primers comp lementary to the untranslated region (UTR) were designed with the help of Primer3 softwa re, except for the pair of primers that were complementary to the exact start and e nd of the gene (LIVUGT_F1 and LIVUGT_R1 respectively). Table 5-6. Primers used for amplifying liver and intestinal UGT gene GSP ID Sequence (5' 3') Start position UTR_F1 CTGCTTCCTCTAGACGTAATTAGAAAC 40 UTR_F2 CTCACATTCCTCCTCCTTCTTTTT 76 UTR_R1 GAACGTGGTGATGAGAACACTATAACT + 121 UTR_R2 TAGTGACATCATAACAACCGTAACTGC + 190 LIVUGT_F1 ATGCCTCGTCTTCTTGCAGCTCTCTGT 1 LIVUGT_R1 TCACTCCTTTTTGCTCTTCTGAGCCCT 1568 Due to the length of the amplicon (~1.6kb), Super Taq Plus polymerase (Ambion Inc) was employed. This enzyme results in higher yields with amplicons >1kb. In addition, this enzyme mixture has a proof-r eading ability, which will be important for future expression of the gene, as well as providing greater fi delity and processivity than ordinary thermos Taq DNA polymerase. An extension temperature of 68°C and an extension time of 1.75 min were used for th is PCR. Different combinations of UTR

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88 primer pairs were tested in order to ensure optimal conditions for amplification of the full-length gene. This experiment was repeated for intestinal cDNA in orde r to investigate whether a similar isoform to that identified from liver is present in the catfish intestine. PCR product purification, ligation and cloning. The procedure followed was identical to the one used for the ini tial study utilizing de generate primers. Samples were submitted to the UF DNA Sequencing Core Facility for DNA sequence analysis. DNA sequencing was carri ed out on both UGT clones as well as products purified from PCR. At least three clones containing the same insert or two separate RACE PCR reactions for PCR amp lification products (comprising 4 sequencing reactions) were sequenced and compared w ith each other for sequence similarity and verification. Bioinformatic analysis of DNA sequences. The DNA sequences obtained were subjected to BLAST search (Alt schul et al., 1997) fo r studying their sim ilarity with other sequences in GenBank. Protein sequences we re predicted by ExpAsy (available online, http://ca.expasy.org/tools/dna .html). Multiple sequence alignments for both nucleotide and predicted amino acid sequences utilized online tools such as ClustalW (available online, http://www.ebi.ac.uk/clustalw/), Basi c Local Alignment Search Tool (BLAST, available online http://www.nc bi.nlm.nih.gov/blast/), as well as BioEdit (downloadable program available from http://www.mbio.ncsu.edu/BioEdit/bioedit.html ). Nucleotide sequence data was analyzed using the B DGP Neural Network Promoter Predictor ( http://www.fruitfly.org/ seq_tools/promoter.html ). Protein sequence data was analyzed by the NCBI Conserved Domain Database Search tool ( http://www.ncbi.nlm.nih.gov/

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89 Structure/cdd/cdd.shtml; Marchler-Bau er and Bryant 2004), Emboss software ( http://www.bioweb.pasteur.fr ) and CLC Protein Workbench v.1.5.2 ( http://www.clcbio. com ). Results (part 2) The RACE studies enabled the successful amplification and cloning of several partial DNA sequences with good similarity to known UGTs and unknown fish sequences. In the case of the liver, partial ov erlapping sequences obt ained via cloning and amplification of PCR products resulted in the recovery of the whole gene (livUGTn, Figure 5-7), including untranslat ed regions at both ends. For the intestine, only partial sequences, corresponding to two distinct is oforms were obtained (I4_3R and I35R_PCR) by RACE. However, the use of GSPs UTR_F1 and UTR_R2 resulted in the amplification and cloning of an identical gene from intes tine (intUGTn), which was identical to the I43R partial sequence. The I35R_PCR sequen ce was only 39% homologous with the full length UGTs obtained from liver and intestine. A comparison of the relative sizes of these sequences with respect to the full-length liver and intestinal UGT genes is shown in Figure 5-8. For both liver and intestine truncated forms of UGT were identified (L25R_5A, I4_6A, I35R_PCR). These lacked the coding sequence for the transmembrane segment at the 3`-end of the gene. The sequences for the amplified, partia l-sequence cDNAs that were sequenced directly or when cloned are given in Appendix A. Nucleotide sequence analysis a. Full-length UGT from liver/intestine. The UGTn sequence was subjected to a BLASTn search (Table 5-7), showing that the sequence exhibite d a high degree of similarity to known UGT sequences. However, DNA sequences are inherently noisy (due

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90 to the 25% probability of a match at any sp ecific position). Cleaner results were obtained by using the predicted protein sequence (Refer to following section). A further advantage of searching at the protein level is that rela ted proteins are generally more conserved than are the genes encoding for them. The GC c ontent was found to be around 50% and a 50 bp long sequence at the 5'-end was predicted to be a possible promoter binding site (Table 5-8), although this may be the promoter of another gene due to its overlap with the translated part of the sequence. 1 GGAATGCTAA GAGCTCGAGT ACCGGGCCTG TTCTTCTCAC ATTCCTCCTC CTTCTTTTTT 61 TCCTCCAAAA TCTGCTTCCT CTAGACGTAA TTAGAAACTT TTAAGCTAAA AATGCCTCGT 121 CTTCTTGCAG CTCTCTGTCT CCAGATTTAT CTTTGCAGCT TTTTAGGACC AGTGGAAGGA 181 GGGAAGGTCC TGGTGATGCC CGTGGACGGC AGCCACTGGC TCAGTATGAA GATCTTGGTG 241 GAGGAATTGT CTCGGAGAGG ACATGAAATG GTGGTCCTGG TTCCCGAGAC AAGCGTGTTG 301 ATCCATGGCT CTGACGCGTA CGCCGCTCGG AGCTTTAAGG TTCCGTACAC CAAGGCTGAA 361 CTGGATGAAA GCATGAATAA GTTGAAGGAG GGCATTACGA AAGCACCGCG GATCTCTGAC 421 TTATTGGAGA ACATCATCGG GCTCCTCAGC TTCACGAACA TGCAGGTGAA AGGATGCGAG 481 GCGCTGCTGT ATAACGAGCC TCTGATGCAG AACCTGCGCG AGGAACACTT CGATCTCATG 541 CTCACCGATC CCTTCCTGCC TTGTGGCCCC ATCATCGCCG AGGCTTTCTC CCTCCCCGCC 601 GTTTATTTCC TGCGTGGGCT TCCCTGCGGA TTGGATCTGG AAGCCGCTCA GTGCCCATCG 661 CCTCCGTCCT ACGTCCCGCG CTTTTTCACA GGCAACACCG ACGTCATGAC GTTTTCTCAG 721 AGGGTCAAGA ACGTGCTCAT GACGGGATTC GAGAGCATCC TTTGCAAAAT ATTTTTCTCC 781 AGCTTTGATG AGCTCACCAG CAGATATCTC AAGAAGGATG TTACGTTCAG AGACGTCCTC 841 GGACATGCCG CGATTTGGCT TTATAGATAT GACTTCACCT TTGAGTACCC GAGACCTGTA 901 ATGCCCAATG CGGTCAGAAT TGGTGGCATC AACTGTGCCA AGAAGAATCC TCTGCCTGCC 961 GATCTGGAGG AGTTCGTGGA CGGTTCTGGA GATCACGGCT TCATCGTGTT CACTTTGGGC 1021 TCCTTCGTGT CCGAGCTGCC GGAGTTCAAA GCCCGGGAGT TTTTCGAGGC TTTTCGGCAG 1081 ATTCCTCAGA GGGTTCTGTG GCGATACACC GGGGTCATTC CCAAAGACAT TCCTGAAAAT 1141 GTCAAAGTGA TGAAGTGGCT TCCGCAGAAC GACCTCTTAG CACACCCCAA GGCTAAGGTG 1201 TTCATCACGC ACGGAGGAGC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG 1261 GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGGA 1321 GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG 1381 AAAGTCCTCA ACAACAAGCG CTACAAAGAG AAGATAACAC AGCTGTCTTT GATCCATAAA 1441 GACCGTCCGA TCGAGCCGCT GGACTTGGCC GTGTTCTGGA CCGAGTTTGT GATGAGACAC 1501 GGAAGTGCCG AGCACCTGAG ACCGGCCGCT CACCACCTCA ACTGGGTTCA GTACCACAGT 1561 CTCGATGTCA TCGCCTTCCT CCTGCTCGTT CTATCCACCG TCGTTTTTAT CGCCGTCAAA 1621 ACCTGCGCGC TCTGTTTCAG GAAGTGTTTC CGGAGGGCTC AGAAGAGCAA AAAGGAGTGA 1681 AACGGCCAGT GAATGATCAG GAATGGATTT GGTGCCGTCT TTAATTAACG CCGATGGTTT 1741 ATCGGCGTGA TGTCATACTG TGAAAACCTG AAATAGTTAT AGTGTTCTCA TCACCACGTT 1801 CAATTTAATA TTCAGGGGTG CCAGCAATTA TGGTTTAGCC ATTGCAGTTA CGGTTGTTAT 1861 GATGTCACTA AAAAAAAAAA A Figure 5-7. Full nucleotide sequence obtaine d for hepatic catfish UGT (livUGTn), derived from 4 sequencing runs each. Highlighted areas indicate start and stop codons (identified after analysis of predicted amino acid sequence).

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91 0 200 400 600 800 1000 1200 1400 livUGTn 5` 3` L25R L25R_4Bb L25R_5A -AAAA -AAAA -AAAA L25R_5R_PCR L7_degenerate I35R I4_degenerate I4_6A -AAAA I4_3R -AAAA bp I35R_PCR -AAAA intUGTn -AAAA Figure 5-8. Sizes and positions of partial UGT sequences (cross-hatched rectangles) from intestine and liver, corresponding to tw o distinct isoforms, relative to complete sequences for liver and inte stinal UGT (solid rectangles). The -AAAA indicates 3 -polyA tail while the suffi x _PCR indicates amplicons that were not cloned but sequenced directly.

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92 Table 5-7. Results of blastn search for livUGTn (and intUGTn) Score E Sequences producing significant alignments: (Bits) Value gi|56324617|emb|CR646752.3|CNS0EY06 Tetraodon nigroviridis full-length cDNA 97.6 1e-16 gi|56242288|emb|CR644097.2|CNS0EVYF Tetraodon nigroviridis full-length cDNA 97.6 1e-16 gi|34850459|dbj|AB120133.1| Pleuronectes yokohamae UGT1B2 mRNA, complete cds 75.8 5e-10 gi|71679708|gb|BC100055.1| Danio rerio cDNA clone IMAGE:7284571, partial cds 71.9 7e-09 gi|68369305|ref|XM_682293.1| PREDICTED: Danio rerio similar to UGT 1, mRNA 71.9 7e-09 gi|68369293|ref|XM_681739.1| PREDICTED: Danio rerio similar to UGT1, mRNA 71.9 7e-09 gi|46518141|emb|BX005348.9| Zebrafish DNA sequence from clone, complete sequence 71.9 7e-09 gi|46016516|emb|BX323548.11| Zebrafish DNA sequence from clone, complete sequence 71.9 7e-09 gi|6537143|gb|AF104339.1|AF104339 Macaca fascicularis UGT1A01 mRNA comp. cds 63.9 2e-06 gi|47087384|ref|NM_213422.1| Danio rerio zgc:66393 (zgc:66393), mRNA, complete cds 60.0 3e-05 gi|33416924|gb|BC055635.1| Danio rerio zgc:66393, mRNA (cDNA) complete cds 60.0 3e-05 gi|50370246|gb|BC075892.1| Danio rerio zgc:66393, mRNA (cDNA) complete cds 60.0 3e-05 gi|62531208|gb|BC093347.1| Danio rerio zgc:66393, mRNA (cDNA) complete cds 60.0 3e-05 gi|32251578|emb|AL954329.7| Zebrafish DNA sequence from clone, complete sequence 60.0 3e-05 gi|81097721|gb|BC109404.1| Danio rerio zgc:123097, mRNA (cDNA) complete cds 60.0 3e-05 gi|82658295|ref|NM_001037428.1| Danio rerio zgc:123097 (zgc:1230), mRNA 60.0 3e-05 gi|50750130|ref|XM_421883.1| PREDICTED: Gallus gallus similar to UGT, mRNA 58.0 1e-04 gi|89572711|gb|AC161471.3| Gallus gallus BAC clone CH261-21B3, complete sequence 58.0 1e-04 gi|46425671|emb|BX931804.2| Gallus gallus finished cDNA, clone ChEST795f19 58.0 1e-04 Table 5-8. Promoter prediction. Predicted transcription start is shown in larger font. Start End Score Promoter Sequence _______________________________________________________________________ 97 147 0.99 AA TTAGAAACTT TTAAGCTAAA AATGCCTCGT CTTCTTGCAGCTCT 480 530 0.98 GAAAGGATGCGAGGCGCTGCTGTATAACGAGCCTCTGATGCAGAACCTGC 1425 1475 0.93 GATAACACAGCTGTCTTTGATCCATAAAGACCGTCCGATCGAGCCGCTGG 1710 1760 0.95 CAGGAATGGATTTGGTGCCGTCTTTAATTAACGCCGATGGTTTATCGGCG Bold sequence indicates most likely promoter The open reading frame (ORF) was identifie d by translating the sequence data of all possible frames (Figure 5-9) and choosi ng the one that showed the least stop codons (Frame +1). The translated sequence (Figure 5-10) was then subjected to a blastp search with other protein sequences in GenBank (T able 5-9), followed by alignment of these sequences. In this way, the untranslated regi ons (UTRs) were also identified (Figure 511). The catfish liver sequence was found to have the best similarity with Danio rerio

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93 UGTs and other unidentified proteins of th e same species (Figure 5-12). The livUGTp sequence was also aligned with mamm alian UGTs (Figures 5-13 and 5-14). Figure 5-9. Identification of open reading frame using ORF Finder 1 GMLRARVPGL FFSHSSSFFF SSKICFL*T* LETFKLKMPR LLAALCLQIY 51 LCSFLGPVEG GKVLVMPVDG SHWLSMKILV EELSRRGHEM VVLVPETSVL 101 IHGSDAYVAR SFKVPYTKAE LDESMNKLKE GITKAPRISD LLENIIGLLS 151 FTNMQVKGCE ALLYNEPLMQ NLREEHFDLM LTDPFLPCGP IIAEAFSLPA 201 VYFLRGLPCG LDLEAAQCPS PPSYVPRFFT GNTDVMTFSQ RVKNVLMTGF 251 ESILCKIFFS SFDELTSRYL KKDVTFRDVL GHAAIWLYRY DFTFEYPRPV 301 MPNAVRIGGI NCAKKNPLPA DLEEFVDGSG DHGFIVFTLG SFVSELPEFK 351 AREFFEAFRQ IPQRVLWRYT GVIPKDIPEN VKVMKWLPQN DLLAHPKAKV 401 FITHGGAHGI YEGICNGVPM VMIPLFGDQV DNVLRMVLRG VAESLTMFDL 451 TSEQLLGALR KVLNNKRYKE KITQLSLIHK DRPIEPLDLA VFWTEFVMRH 501 GSAEHLRPAA HHLNWVQYHS LDVIAFLLLV LSTVVFIAVK TCALCFRKCF 551 RRAQKSKKE* NGQ*MIRNGF GAVFN*RRWF IGVMSYCENL K*L*CSHHHV 601 QFNIQGCQQL WFSHCSYGCY DVTKKKK Figure 5-10. Predicted protein seque nce liv/intUGTp from liv/intUGTn * indicate stop codons; highlighted resi dues indicate start and stop residues, determined after comparison with kn own UGT sequences, see Figure 5-11 below)

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94 Table 5-9. Results of blastp search for liv/intUGTp Score E Sequences producing significant alignments: (Bits) Value gi|47087385|ref|NP_998587.1| hypothetical protein LOC406731 [ Danio rerio ] 725 0.0 gi|81097722|gb|AAI09405.1| Hypothetical protein LOC641488 [ Danio rerio ] 692 0.0 gi|50370247|gb|AAH75892.1| Zgc:66393 protein [ Danio rerio ] 691 0.0 gi|71679709|gb|AAI00056.1| Unknown (protein for IMAGE:7284571) [ Danio rerio ] 671 0.0 gi|47205148|emb|CAG04937.1| unnamed protein product [ Tetraodon nigroviridis ] 620 6e-176 gi|34850460|dbj|BAC87829.1| UDP-glucuronosyltransferase [ Pleuronectes yokohamae ] 602 2e-170 gi|5579028|emb|CAB51368.1| UDP-glucuronosyltransferase [ Pleuronectes platessa ] 601 3e-170 gi|6272259|emb|CAB51369.2| UDP-glucuronosyltransferase [ Pleuronectes platessa ] 598 2e-169 gi|62531209|gb|AAH93347.1| Zgc:66393 protein [ Danio rerio ] 580 8e-164 gi|47210873|emb|CAF91810.1| unnamed protein product [ Tetraodon nigroviridis ] 573 8e-162 gi|68369306|ref|XP_687385.1| PREDICTED: similar to UGT1 family [ Danio rerio ] 570 5e-161 gi|68369294|ref|XP_686831.1| PREDICTED: similar to UGT1 family [ Danio rerio ] 568 3e-160 gi|2842546|dbj|BAA24692.1| UDP-glucuronosyltransferase [ Felis catus ] 555 2e-156 gi|57163903|ref|NP_001009359.1| UDP glycosyltransferase 1A1 [ Felis catus ] 554 4e-156 gi|56785765|gb|AAW29020.1| UDP-glucuronosyltransferase [ Epinephelus coioides ] 547 6e-154 gi|975702|emb|CAA52214.1| UDP-glucoronosyl transferase [ Pleuronectes platessa ] 544 4e-153 gi|40849838|gb|AAR95631.1| UGT1A2 [ Rattus norvegicus ] 542 2e-152 gi|40849834|gb|AAR95629.1| UGT1A1 [ Rattus norvegicus ] 541 3e-152 gi|207579|gb|AAA42312.1| bilirubin UDP-gluc uronosyltransferase [ Rattus norvegicus ] 541 3e-152 gi|2507507|sp|P20720|UD12_RAT UGT 1-2 precursor [ Rattus norvegicus ] 541 3e-152 gi|46518737|gb|AAS99732.1| UGT1A1 [ Homo sapiens ] 536 1e-150 gi|40849842|gb|AAR95633.1| UGT1A6 [ Rattus norvegicus ] 535 3e-150 gi|62533164|gb|AAH93516.1| UGT1A1 [ Mus musculus ] 535 3e-150 gi|89519335|gb|ABD75811.1| UDP glycosyl transferase 1A1 [ Papio anubis ] 535 3e-150 gi|8170744|gb|AAB26033.2| UDP-glucuronosyltransferase [ Mus sp. ] 534 4e-150 gi|6537144|gb|AAF15549.1| UGT1A01 [ Macaca fascicularis ] 534 5e-150 gi|74136303|ref|NP_001028041.1| UGT1A01 [ Macaca mulatta ] 533 7e-150 gi|2501477|sp|Q64638|UD15_RAT UGT1A5 precursor [ Rattus norvegicus ] 533 7e-150

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95 Scores SeqA Name Len(aa) SeqB Name Len(aa) Score ========================= ==================== ================== 1 livUGTp 627 2 D.rerioPR 526 67 1 livUGTp 627 3 P.platessaUGT 530 54 1 livUGTp 627 4 T.nigroviridisPR 501 59 1 livUGTp 627 5 P.yokohamaeUGT 530 54 2 D.rerioPR 526 3 P.platessaUGT 530 54 2 D.rerioPR 526 4 T.nigroviridisPR 501 60 2 D.rerioPR 526 5 P.yokohamaeUGT 530 55 3 P.platessaUGT 530 4 T.nigroviridisPR 501 60 3 P.platessaUGT 530 5 P.yokohamaeUGT 530 91 4 T.nigroviridisPR 501 5 P.yokohamaeUGT 530 60 ========================= ==================== ================== Accession numbers: D.rerioPR (N P_998587.1); P.platessaUGT (CAB51368.1); T.nigroviridisPR (CAG04937.1); P.yokohamaeUGT (BAC87829.1) CLUSTAL W (1.83) multiple sequence alignment P.platessaUGT -----------------------------------MSGRVWFQMLGLVAWLCLLSLGPVQ 25 P.yokohamaeUGT -----------------------------------MSGRVWFPSLGLVAWLCLLSLGPVQ 25 T.nigroviridisPR -----------------------------------MRGSVGILTLGLLAWIGCFGPQPVQ 25 livUGTp GMLRARVPGLFFSHSSSFFFSSKICFL-T-LETFKLKMPRLLAALCLQIYLCSFLG-PVE 57 D.rerioPR -----------------------------------MKRTLFVPALGLFAFLCLFSSESVQ 25 : . * * :: : .*: P.platessaUGT GGKVLVMPADGSHWLSLKILVKGLIHRGHDVVVLVPESSLFMHQSEDYKTEVYPVSFTTE 85 P.yokohamaeUGT GGKVLVMPADGSHWLSLKILVKGLVHRGHDVVVLVPESSLFMHQSEDYKTEVYPVSFTME 85 T.nigroviridisPR AGKVLVLPVDGSHWLSMKILVKELIQRGHDVLVLVPETSLLIKSSENYRTEIYQVPYSKE 85 livUGTp GGKVLVMPVDGSHWLSMKILVEELSRRGHEMVVLVPETSVLIHGSDAYVARSFKVPYTKA 117 D.rerioPR AGKVLVMPVDGSHWLSMKILVEEMSSRGHEMVVLVPETSILIGKSGNFTTKSFRVPYSFD 85 .*****:*.*******:****: : ***:::*****:*::: * : :. : *.:: P.platessaUGT EMDATHKQLKDGLFLKQPDWTEYYVNIMRFVNFTSIHLRGCENLLENQPLMSRMRGMGFD 145 P.yokohamaeUGT EMDAVHKQLKDGLFLKQPDWTEYYVNIMRFVNFTSIHLRGCENLLQNQPLMSRLKGMGFD 145 T.nigroviridisPR DLGGSFQALKDGLFLKPPSMADLFVNVERLMNFTTMQVTGCESLLRNQPLMTRLREQGFE 145 livUGTp ELDESMNKL-KEGITKAPRISDLLENIIGLLSFTNMQVKGCEALLYNEPLMQNLREEHFD 176 D.rerioPR ELNAHVDHIRKTAIEKAPRFIDIVGALGNLIQFTNMQVKACEGLLYDEPLMKSLRDMKFD 145 ::. . : . : * * : : ::.**.::: .** ** ::*** :: *: P.platessaUGT IVLTDPFFPCGALVGNIFSIPVVNFLRGLPCGLDMKVNKCPSPPSYIPVPYSGNTNIMTF 205 P.yokohamaeUGT IVLTDPFFPCGALVGHIFSIPVVNFLRGLPCGLDMKVNKCPSPPSYIPVPYSGHTDIMTF 205 T.nigroviridisPR VVLTDPFLPCGPIVSHLFNIPAVYFLHGLPCELDSKANQCPAPPSYIPTSFSGNSDVMTF 205 livUGTp LMLTDPFLPCGPIIAEAFSLPAVYFLRGLPCGLDLEAAQCPSPPSYVPRFFTGNTDVMTF 236 D.rerioPR ALLTDPFLPCGSVIADYFSIPAVYFLRGIPCRLDEAAAQCPSPPSFIPRFFTGYTDKMTF 205 :*****:***.::.. *.:*.* **:*:** ** . :**:***::* ::* :: *** P.platessaUGT PQRVINMAMTVLESYQCSLLYGHYDEMVSKYVGNNMDYRTLLSHGALWLIRNEFTLDWAR 265 P.yokohamaeUGT QQRVINMAMTVVESFQCSLLYSHYDEMVSKHLGNNMDYRTLLSNGALWLIRNEFSLDWPR 265 T.nigroviridisPR PQRVKNMLMYLVQSYLCKVMYREFDRLVTRHMSDIQSYRELISRGAFWLLKYDFTFQHPK 265 livUGTp SQRVKNVLMTGFESILCKIFFSSFDELTSRYLKKDVTFRDVLGHAAIWLYRYDFTFEYPR 296 D.rerioPR PQRMINTFMTVFEKYLCHQLFASFDELATRYLKKDTSYAELLGHGAVWLLRYDFSFEYPK 265 **: * * .:. :* :: :*.:.:::: . : ::...*.** : :*::: .: P.platessaUGT PLLPNMVLIGGINCAEKKKNASLPADLEEFVQGSGDDGFIIFTLGSMLPDMPQEKAQHFL 325 P.yokohamaeUGT PLLPNMVLIGGINCAEKKTKASLPADLEEFVQGSGDHGFIIFTLGSMLPDMPQEMAQHFL 325 T.nigroviridisPR PVMPNTAFIGGINCAKK---APLPADLEEFVNGSEDHGFIVFSLGSMVENMPVEKAKQFF 322 livUGTp PVMPNAVRIGGINCAKK---NPLPADLEEFVDGSGDHGFIVFTLGSFVSELPEFKAREFF 353 D.rerioPR PQMPNMVQIGGINCAKR---APLTKELEEFVNGSGEHGFVVFTLGSMVSQLPEAKAREFF 322 * :** . *******:: .*. :*****:** :.**::*:***:: ::* *:.*: Figure 5-11. Comparison of liv/intUGTp w ith homologous proteins in other fish, showing scores and alignment of closely related sequences.

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96 P.platessaUGT DAFRQIPQRVVWRYAGDPPKGLPKNVRLMKWLPQKELLAHPKARLFLTHGGSHSVYEGIC 385 P.yokohamaeUGT DAFRQIPQRVVWRYAGEPPKGLPKNVKLMKWLPQKDLLAHPKAKLFLTHGGSHSVFEGIC 385 T.nigroviridisPR DAFAQIPQRVLWRYNGAVPENAPKNVKVMKWLPQNDLLAHPKAKVFMTHGGIHGIYEGIC 382 livUGTp EAFRQIPQRVLWRYTGVIPKDIPENVKVMKWLPQNDLLAHPKAKVFITHGGAHGIYEGIC 412 D.rerioPR EAFRQIPQRVLWRYTGPVPENAPKNVKLMKWLPQNDLLGHPKVRAFVTHGGSHGIYEGIC 382 :** ******:*** * *:. *:**::******::**.***.: *:**** *.::**** P.platessaUGT NAVPMLMFPLFAEQGDNGLRMVTRGAAETLNIYDVTSDNLLAALNKILKNKSYKEKITEM 445 P.yokohamaeUGT NAVPMLMFPLFAEQGDNGLRMVTRGVAETLFIYDVTSDTLLATLNKILKNKSYKEKMTEL 445 T.nigroviridisPR NGVPMLMFPLFGDQIDNVPRMIHRGVAETLSIYDVTSQKLVAALKKMVQDKSYKENMVTL 442 livUGTp NGVPMVMIPLFGDQVDNVLRMVLRGVAESLTMFDLTSEQLLGALRKVLNNKRYKEKITQL 472 D.rerioPR NGVPMVMLPLFGDQGDNAQRLVSRGVAESLTIYDVTSEKLLVALKKVINDKSYKEKMMKL 442 *.***:*:***.:* ** *:: **.**:* ::*:**: *: :*.*::::* ***:: : P.platessaUGT SQIHHDRPVAPLDLAVFWTEFVIRHKGASHLRVAAHELNWIQYHSLDVFGFILLILLTVL 505 P.yokohamaeUGT SQIHHDRPVGPLDLAIFWTEFVIRHKGAAHLRVSAHDLNWIQYHSLDVFGFLLLILLTVL 505 T.nigroviridisPR SQLNQDRPVAPLDLAVFWTEFVMRHQGAQHLRVPPHDLNWFQYHSLDIFCFLAVVLLTV501 livUGTp SLIHKDRPIEPLDLAVFWTEFVMRHGSAEHLRPAAHHLNWVQYHSLDVIAFLLLVLSTVV 532 D.rerioPR SAIHRDRPIEPLDLAVFWTEFVMRHKGAEHLRPAAHDLNWIQYHSLDVIGFLLLILLTVI 502 * :::***: *****:******:** .* *** ..*.***.******:: *: ::* ** P.platessaUGT WATLKCCLFCTRRCCRRGTAKTKSE----------------------------------530 P.yokohamaeUGT LVTLKCCLSCTRRCCRRGTAKTKSE----------------------------------530 T.nigroviridisPR -----------------------------------------------------------livUGTp FIAVKTCALCFRKCFRRAQKSKKE-NGQ-MIRNGFGAVFN-RRWFIGVMSYCENLK-L-C 587 D.rerioPR FVTVKSCMFCFRKCFKTSQKKKKA-----------------------------------526 P.platessaUGT -------------------------------P.yokohamaeUGT -------------------------------T.nigroviridisPR -------------------------------livUGTp SHHHVQFNIQGCQQLWFSHCSYGCYDVTKKKK 619 D.rerioPR -------------------------------Figure 5-11. (continued) Highlighted area in livUGTp indicates UTRs. Figure 5-12. Phylogram for fish UGT proteins homologous to liv/intUGTp

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97 Scores SeqA Name Len(aa) SeqB Name Len(aa) Score ========================= ==================== ================== 1 I.punctatus_livUGTp 522 2 F.catus_UGT1A1 533 51 1 I.punctatus_livUGTp 522 3 R.norvegicus1A2 533 51 1 I.punctatus_livUGTp 522 4 R.norvegicus1A1 535 50 1 I.punctatus_livUGTp 522 5 H.sapiensUGT1A1 533 48 1 I.punctatus_livUGTp 522 6 R.norvegicus1A6 531 51 2 F.catus_UGT1A1 533 3 R.norvegicus1A2 533 67 2 F.catus_UGT1A1 533 4 R.norvegicus1A1 535 76 2 F.catus_UGT1A1 533 5 H.sapiensUGT1A1 533 79 2 F.catus_UGT1A1 533 6 R.norvegicus1A6 531 67 3 R.norvegicus1A2 533 4 R.norvegicus1A1 535 70 3 R.norvegicus1A2 533 5 H.sapiensUGT1A1 533 66 3 R.norvegicus1A2 533 6 R.norvegicus1A6 531 88 4 R.norvegicus1A1 535 5 H.sapiensUGT1A1 533 78 4 R.norvegicus1A1 535 6 R.norvegicus1A6 531 70 5 H.sapiensUGT1A1 533 6 R.norvegicus1A6 531 67 Accession numbers: F.catusUGT1A 1 (NP_001009359.1); R.norvegicusUGT1A2 (AAR95631.1); R.norvegicus1A1 (AAR 95629.1); R.norvegicus1A6 (AAR95633.1); H.sapiensUGT1A1 (AAS99732.1) F.catus_UGT1A1 MAARSRGPRPLVLS--LLLCALNPLLSQGGKLLLVPMDGSHWLSLFGVIQRLHQRGHDVV 58 H.sapiensUGT1A1 MAVESQGGRPLVLG--LLLCVLGPVVSHAGKILLIPVDGSHWLSMLGAIQQLQQRGHEIV 58 R.norvegicus1A1 MSVVCRSSCSLLLLPCLLLCVLGPSASHAGKLLVIPIDGSHWLSMLGVIQQLQQKGHEVV 60 R.norvegicus1A2 MDTGLCAPLRGLSGLLLLLCALP--WAEGGKVLVFPMEGSHWLSMRDVVRELHARGHQAV 58 R.norvegicus1A6 --MGLHVTLQGLAGLLLLLYALP--WAEGGKVLVFPMEGSHWLSMRDVVRELHARGHQAV 56 I.punctatus_livUGTp -----MPRLLAALCLQIYLCSFLG-PVEGGKVLVMPVDGSHWLSMKILVEELSRRGHEMV 54 : * : ..**:*:.*::******: :..* :**: * F.catus_UGT1A1 VVAPEASVYIKEGAFYTLKSYPVPFRREDVEASFTGLGLGVFEKKPFLQRVVATYKRVKK 118 H.sapiensUGT1A1 VLAPDASLYIRDGAFYTLKTYPVPFQREDVKESFVSLGHNVFENDSFLQRVIKTYKKIKK 118 R.norvegicus1A1 VIAPEASIHIKEGSFYTMRKYPVPFQNENVTAAFVELGRSVFDQDPFLLRVVKTYNKVKR 120 R.norvegicus1A2 VLAPEVTVHMKGEDFFTLQTYAFPYTKEEYQREILGNAKKGFEPQHFVKTFFETMASIKK 118 R.norvegicus1A6 VLAPEVTVHIKEEDFFTLQTYPVPYTRQGFRQQMMRNIKVVFETGNYVKTFLETSEILKN 116 I.punctatus_livUGTp VLVPETSVLIHGSDAYVARSFKVPYTKAELDESMNKLKEGIT-KAPRISDLLENIIGLLS 113 *:.*:.:: :: :: :.: .*: . : : .. . : F.catus_UGT1A1 DSALLLSACSHLLYNEELMASLAESGFDAMLTDPFLPCGPIVALRLALPVVFFLNSLPCG 178 H.sapiensUGT1A1 DSAMLLSGCSHLLHNKELMASLAESSFDVMLTDPFLPCSPIVAQYLSLPTVFFLHALPCS 178 R.norvegicus1A1 DSSMLLSGCSHLLHNAEFMASLEQSHFDALLTDPFLPCGSIVAQYLSLPAVYFLNALPCS 180 R.norvegicus1A2 FFDLYANSCAALLHNKTLIQQLNSSSFDVVLTDPVFPCGALLAKYLQIPAVFFLRSVPCG 178 R.norvegicus1A6 ISTVLLRSCMNLLHNGSLLQHLNSSSFDMVLTDPVIPCGAVLAKYLGIPTVFFLRYIPCG 176 I.punctatus_livUGTp FTNMQVKGCEALLYNEPLMQNLREEHFDLMLTDPFLPCGPIIAEAFSLPAVYFLRGLPCG 173 : .* **:* :: * .. ** :****.:**..::* : :*.*:**. :**. F.catus_UGT1A1 LDFQGTRCPSPPSYVPRVLSLNSDHMTFLQRVKNMLILGSEGFLCNVVYSPYASLASEVL 238 H.sapiensUGT1A1 LEFEATQCPNPFSYVPRPLSSHSDHMTFLQRVKNMLIAFSQNFLCDVVYSPYATLASEFL 238 R.norvegicus1A1 LDLEATQCPAPLSYVPKSLSSNTDRMNFLQRVKNMIIALTENFLCRVVYSPYGSLATEIL 240 R.norvegicus1A2 IDYEATQCPKPSSYIPNLLTMLSDHMTFLQRVKNMLYPLTLKYICHLSITPYESLASELL 238 R.norvegicus1A6 IDSEATQCPKPSSYIPNLLTMLSDHMTFLQRVKNMLYPLALKYICHFSFTRYESLASELL 236 I.punctatus_livUGTp LDLEAAQCPSPPSYVPRFFTGNTDVMTFSQRVKNVLMTGFESILCKIFFSSFDELTSRYL 233 :: :.::** * **:*. :: :* *.* *****:: :* . : : *::. * Figure 5-13. Alignment of liv/intUGTp (e xcluding UTRs) with selected mammalian UGT proteins, showing scores and multiple alignment of sequences, highlighting important regions and residues (see discussion)

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98 F.catus_UGT1A1 QKDVTVQDLMGSASVWLFRSDFVKDYSRPIMPNMVFIGGINCAGKNPLSQEFEAYVNASG 298 H.sapiensUGT1A1 QREVTVQDLLSSASVWLFRSDFVKDYPRPIMPNMVFVGGINCLHQNPLSQEFEAYINASG 298 R.norvegicus1A1 QKEVTVKDLLSPASIWLMRNDFVKDYPRPIMPNMVFIGGINCLQKKALSQEFEAYVNASG 300 R.norvegicus1A2 QREMSLVEVLSHASVWLFRGDFVFDYPRPIMPNMVFIGGINCVIKKPLSQEFEAYVNASG 298 R.norvegicus1A6 QREVSLVEVLSHASVWLFRGDFVFDYPRPVMPNMVFIGGINCVIKKPLSQEFEAYVNASG 296 I.punctatus_livUGTp KKDVTFRDVLGHAAIWLYRYDFTFEYPRPVMPNAVRIGGINCAKKNPLPADLEEFVDGSG 293 :::::. :::. *::** * **. :*.**:*** * :***** ::.*. ::* :::.** F.catus_UGT1A1 EHGIVVFSLGSMVSAIPKEKAMEIADALGKIPQTVLWRYTGTPPPNLAKNTILVKWLPQN 358 H.sapiensUGT1A1 EHGIVVFSLGSMVSEIPEKKAMAIADALGKIPQTVLWRYTGTRPSNLANNTILVKWLPQN 358 R.norvegicus1A1 EHGIVVFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTRPSNLAKNTILVKWLPQN 360 R.norvegicus1A2 EHGIVVFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTRPSNLAKNTILVKWLPQN 358 R.norvegicus1A6 EHGIVVFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTRPSNLAKNTILVKWLPQN 356 I.punctatus_livUGTp DHGFIVFTLGSFVSELPEFKAREFFEAFRQIPQRVLWRYTGVIPKDIPENVKVMKWLPQN 352 :**::**:***:** :*: ** : :*: :*** *******. * ::.:*..:::***** ---F.catus_UGT1A1 DLLGHPKARAFITHSGSHGIYEGICNGVPMVMLPLFGDQMDNAKRMETRGAGLTLNVLEM 418 H.sapiensUGT1A1 DLLGHPMTRAFITHAGSHGVYESICNGVPMVMMPLFGDQMDNAKRMETKGAGVTLNVLEM 418 R.norvegicus1A1 DLLGHPKARAFITHSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMETRGAGVTLNVLEM 420 R.norvegicus1A2 DLLGHPKARAFITHSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMETRGAGVTLNVLEM 418 R.norvegicus1A6 DLLGHPKARAFITHSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMETRGAGVTLNVLEM 416 I.punctatus_livUGTp DLLAHPKAKVFITHGGAHGIYEGICNGVPMVMIPLFGDQVDNVLRMVLRGVAESLTMFDL 412 ***.** ::.****.*:**:**.*********:******:**. ** :*.. :*.:::: ---UDPGA binding site -----------------> F.catus_UGT1A1 TSEDLANGLKAVINDKSYKENIMRLSSLHKDRPIEPLDLAVFWVEFVMRHKGAPHLRPAA 478 H.sapiensUGT1A1 TSEDLENALKAVINDKSYKENIMRLSSLHKDRPVEPLDLAVFWVEFVMRHKGAPHLRPAA 478 R.norvegicus1A1 TADDLENALKTVINNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPAA 480 R.norvegicus1A2 TADDLENALKTVINNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPAA 478 R.norvegicus1A6 TADDLENALKTVINNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPAA 476 I.punctatus_livUGTp TSEQLLGALRKVLNNKRYKEKITQLSLIHKDRPIEPLDLAVFWTEFVMRHGSAEHLRPAA 472 *:::* ..*: *:*:* ***:* :** :*****:*********.*:**** .* ****** F.catus_UGT1A1 HDLTWYQYHSVDVIGFLLAIVLGIVFITYKCCAFGCRKCFGRKGRVKKSHKSKTH 533 H.sapiensUGT1A1 HDLTWYQYHSLDVIGFLLAVVLTVAFITFKCCAYGYRKCLGKKGRVKKAHKSKTH 533 R.norvegicus1A1 HDLTWYQYHSLDVIGFLLAIVLTVVFIVYKSCAYGCRKCFGGKGRVKKSHKSKTH 535 R.norvegicus1A2 HDLTWYQYHSLDVIGFLLAIVLTVVFIVYKSCAYGCRKCFGGKGRVKKSHKSKTH 533 R.norvegicus1A6 HDLTWYQYHSLDVIGFLLAIVLTVVFIVYKSCAYGCRKCFGGKGRVKKSHKSKTH 531 I.punctatus_livUGTp HHLNWVQYHSLDVIAFLLLVLSTVVFIAVKTCALCFRKCFR---RAQKSKKE--521 *.*.* ****:***.*** :: :.**. * ** ***: *.:*::*. TM region Figure 5-13. (continued) Figure 5-14. Phylogram for I.punctatus liv/intUGTp and selected mammalian UGT proteins b. Distinct partial sequence found in intestine (I35R_C). The partial nucleotide sequence of I35R_C (combined overlapping sequences of I35R and I35R_PCR) was subjected to a blastN search (Table 5-10) and shown to have homology with other UGTs and unknown fish cDNAs. Multiple sequence al ignment showed that livUGTn and I4_3R are similar with respect to each other, while I35R was markedly different (Figure 5-15).

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99 Table 5-10. Results of blastn search for I35R_C Score E Sequences producing significant alignments: (Bits) Value gi|56324617|emb|CR646752.3|CNS0EY06 Tetraodon nigroviridis full-length DNA 91.7 2e-15 gi|56242288|emb|CR644097.2|CNS0EVYF Tetraodon nigroviridis full-length DNA 91.7 2e-15 gi|34850459|dbj|AB120133.1| Pleuronectes yokohamae UGT1B2 mRNA 69.9 9e-09 gi|71679708|gb|BC100055.1| Danio rerio cDNA clone IMAGE:7284571, partial cd 60.0 8e-06 gi|46518141|emb|BX005348.9| Zebrafish DNA sequence from clone... 60.0 8e-06 gi|46016516|emb|BX323548.11| Zebrafish DNA sequence from clon... 60.0 8e-06 gi|68369305|ref|XM_682293.1| PREDICTED: Danio rerio similar to UGT1, mRNA 60.0 8e-06 gi|68369293|ref|XM_681739.1| PREDICTED: Danio rerio similar to UGT1, mRNA 60.0 8e-06 Scores SeqA Name Len(nt) SeqB Name Len(nt) Score ================== ================== ================= 1 livUGTn 1881 2 I35R_C 581 39 ================== ================== ================= livUGTn AGCTTTGATGAGCTCACCAGCAGATATCTCAAGAAGGATGTTACGTTCAGAGACGTCCTC 840 I35R_C --------------------------------------------------AAATTCCCAA 10 * * ** livUGTn GGACATGCCGCGATTTGGCTTTATAGATATGACTTCACCTTTGAGTACCCGAGACCTGTA 900 I35R_C AGACATTCCTGAAAATG---TCAAAGTGATGAAGTGGCTTCCGCAGAATGACCTCTTAGG 67 **** ** * ** * * ** **** * * * * * * * livUGTn ATGCCCAATGCGGTCAGAATTGGTGGCATCAACTGTGCCAAGAAGAATCCTCTGCCTGCC 960 I35R_C TTTGTTTACACGTCCTCTAACCGTAATAAATAGACACCCGGTCCCCATTTTCTCTCTCAC 127 * * ** * * ** * * ** ** *** ** * livUGTn GATCTGGAGGAGTTCGTGGACGGTTCTGGAGATCACGGCTTCATCGTGTTCACTTTGGGC 1020 I35R_C ACACACATCTATCTATCACACAGGTCTATGATTATCGATTATACCGTA--CGTTTCCAGC 185 * * ** * ** * ** * * *** * ** ** livUGTn TCCTTCGTGTCCGAGCTGCCGGAGTTCAAAGCCCG----GGAGTTTTTCGAGGCTTTTCG 1076 I35R_C TAA--CACTACTTGGATACTTTGGTCAAAAACTCACACCGAAGGTCATTAACACA---CA 240 * * * * * * ** *** * * * ** * * * * * livUGTn GCAGATTCCTCAGAGGGTTCTGTGGCGATACACCGGGGTCATTCCCAAAGACATTCCTGA 1136 I35R_C GTTCCTGTTTTAAACAGCGTTAAAATT-TAAATCTGAAAGATTCGAGGAAATATAATGGT 299 * * * * * * * ** * * * **** * * ** * livUGTn AAATGTCTA--AGTGATGAGGTGGCTTCCGCAGAACGACCTCTTA----GCACACCCCAA 1190 I35R_C GCATAATAATAATTTCCTTTTTTCTTTCCTTTCATCGCCGTGTTAAAAAGCACACCCCAA 359 ** * * * * **** * ** * * *** *********** livUGTn GGCTAAGGTGTTCATCACGCACGGAGGAGCCCATGGCATCTACGAGGGTATCTGTAACGG 1250 I35R_C GGCTAAGGTGTTCATCACGCACGGAGGAACCCATGGCATCTACGAGGGTATCTGTAACGG 419 **************************** ******************************* livUGTn CGTGCCGATGGTGATGATCCCGCTGTTCGGAGATCAGGTAGACAACGTTCTACGCATGGT 1310 I35R_C CGTGCCGATGGTGATGATCCCGCTGTTCGGAGATCAGGTAGACAACGTTCTACGCATGGT 479 ************************************************************ livUGTn GCTGCGTGGAGTCGCAGAGAGCCTGACCATGTTCGACCTGACCTCAGAGCAACTGCTGGG 1370 I35R_C GCTGCGTGAAGTCGCAGAGAGCCTGACCATGTTCGACCTGACCTCAGAGCAACTGCTGGG 539 ******** *************************************************** livUGTn GGCACTCAGGAAAGTCCTCAACAACAAGCGCTACAAAGAGAAGATAACACAGCTGTCTTT 1430 I35R_C GGCACTCAGGAAAGTCCTCAACAACGAGCGCTAAAAAAAAAA-----------------581 ************************* ******* *** * ** Figure 5-15. Multiple sequence alignment between livUGTn and I35R_C. Part of livUGTn sequence omitted for clarity.

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100 Protein sequence analysis a.liv/intUGTp. Further confirmation that the predicted livUGTp is a UGT came from a conserved domain search (Figure 516). The sequence wa s 100% aligned with UDPGT, and it was also closely related to gl ycosyltransferases and N-acetylglucosamine transferases. Figure 5-16. Results of NCBI conserved domain search PSSM: position-specific scoring matrix The protein displayed severa l areas of marked hydrophobicity , particularly at the Cterminal end, which presumably corresponds to the transmembrane region, which is followed by the positively charged cytoplas mic tail (Figure 5-17 ). Based upon the method developed by Kolaskar and Tongaonka r (1990), the Emboss program antigenic identified several potential antigenic site s. Maximum score position is denoted by the residues in bold and underli ned format (Table 5-11).

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101 Figure 5-17. Kyte-Doolittle Hydroph obicity Plot for liv/intUGTp Table 5-11. Potential antige nic sites on liv/intUGTp. Sequence Start Length Score LLAALCLQ I YLCSFLGPV 4 18 1.236 AEHLRPAAHHLNWVQYHSLDVIAFLLLVL S TVVFIAVKTCALCFRKCFRR 466 50 1.224 DLMLTDPFLPCGPIIAEAFSLPA V YFLRGLPCGLDLEAAQCPSPPSYVPRF 141 51 1.180 EMVVL V PETSVLIHGSDAYVARSFKVPYTKA 52 31 1.175 GGKVL V MPVDGSH 23 13 1.174 ESILC K IFFSSF 214 12 1.154 IEPLD L AVFWTE 447 12 1.138 FRDVLG H AAIWLYRYD 239 16 1.127 VKGCE A LLYNE 119 11 1.123 VDNVLR M VLRGVAESL 393 16 1.12 LSM K ILVEEL 37 10 1.115

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102 B. I35R_Cp. The predicted protei n sequence for the partial cDNA obtained from intestine also exhibited homology with UGT proteins (Table 5-12). Conserved domain search showed that both sequences ali gned with UDPGA-binding dom ains (Figure 5-18). Table 5-12. Results of blastp search for I35R_Cp Score E Sequences producing significant alignments: (Bits) Value gi|47087385|ref|NP_998587.1| hypothetical protein LOC406731 [ Danio rerio ] 114 1e-24 gi|50370247|gb|AAH75892.1| Zgc:66393 protein [ Danio rerio ] 114 1e-24 gi|62531209|gb|AAH93347.1| Zgc:66393 protein [ Danio rerio ] 114 1e-24 gi|81097722|gb|AAI09405.1| Hypothetical protein LOC641488 [ Danio rerio ] 114 1e-24 gi|47205148|emb|CAG04937.1| unnamed protein product [ Tetraodon nigroviridis ] 113 4e-24 gi|47191630|emb|CAF92264.1| unnamed protein product [ Tetraodon nigroviridis ] 112 9e-24 gi|47198340|emb|CAF87158.1| unnamed protein product [ Tetraodon nigroviridis ] 112 9e-24 gi|47197196|emb|CAF89118.1| unnamed protein product [ Tetraodon nigroviridis ] 112 9e-24 gi|47210873|emb|CAF91810.1| unnamed protein product [ Tetraodon nigroviridis ] 111 2e-23 gi|56785765|gb|AAW29020.1| UGT [ Epinephelus coioides ] 108 1e-22 gi|975702|emb|CAA52214.1| UGT [ Pleuronectes platessa ] 105 1e-21 gi|71679709|gb|AAI00056.1| Unknown (protein for IMAGE:7284571)[ Danio rerio ] 104 2e-21 gi|68369306|ref|XP_687385.1| PREDICTED: similar to UGT1 family [ Danio rerio ] 104 2e-21 gi|68369294|ref|XP_686831.1| PREDICTED: similar to UGT1 family [ Danio rerio ] 104 2e-21 gi|5579028|emb|CAB51368.1| UGT [ Pleuronectes platessa ] 103 2e-21 gi|34850460|dbj|BAC87829.1| UGT [ Pleuronectes yokohamae ] 102 7e-21 gi|6272259|emb|CAB51369.2| UGT [ Pleuronectes platessa] 102 9e-21 gi|2842546|dbj|BAA24692.1| UGT [ Felis catus ] 100 3e-20 gi|51260641|gb|AAH78732.1| Ugt1a7 protein [ Rattus norvegicus ] 99.0 8e-20 gi|40849836|gb|AAR95630.1| UGT1A11 [ Rattus norvegicus ] 99.0 8e-20 gi|40849840|gb|AAR95632.1| UGT1A4 [ Rattus norvegicus ] 99.0 8e-20 gi|40849848|gb|AAR95636.1| UGT1A9 [ Rattus norvegicus ] 99.0 8e-20 gi|40849846|gb|AAR95635.1| UGT1A8 [ Rattus norvegicus ] 99.0 8e-20 gi|79160160|gb|AAI07932.1| UGT1A6 [ Rattus norvegicus ] 99.0 8e-20 gi|40849842|gb|AAR95633.1| UGT1A6 [ Rattus norvegicus ] 99.0 8e-20 gi|40849838|gb|AAR95631.1| UGT1A2 [ Rattus norvegicus ] 99.0 8e-20 gi|18308176|gb|AAL67854.1| UGT1A7 [ Rattus norvegicus ] 99.0 8e-20 gi|18308170|gb|AAL67851.1| UGT1A8 [ Rattus norvegicus ] 99.0 8e-20 gi|18308168|gb|AAL67850.1| UGT1A5 [ Rattus norvegicus ] 99.0 8e-20 gi|18308174|gb|AAL67853.1| UGT1A6 [ Rattus norvegicus ] 99.0 8e-20 gi|207579|gb|AAA42312.1| bilirubin UGT [ Rattus norvegicus ] 99.0 8e-20 gi|136726|sp|P08430|UD16_RAT UGT 1-6 [ Rattus norvegicus ] 99.0 8e-20 gi|40849834|gb|AAR95629.1| UGT1A1 [ Rattus norvegicus ] 99.0 8e-20 gi|2501482|sp|Q64634|UD18_RAT UDP-glucuronosyltransferase 1-8... 99.0 8e-20 gi|2501481|sp|Q64633|UD17_RAT UDP-glucuronosyltransferase 1-7... 99.0 8e-20 gi|2501477|sp|Q64638|UD15_RAT UDP-glucuronosyltransferase 1-5... 99.0 8e-20 gi|2501475|sp|Q64637|UD13_RAT UDP-glucuronosyltransferase 1-3... 99.0 8e-20 gi|2507507|sp|P20720|UD12_RAT UDP-glucuronosyltransferase 1-2... 99.0 8e-20 gi|57163923|ref|NP_001009383.1| UGT1A [ Felis catus ] 98.6 1e-19 gi|57163903|ref|NP_001009359.1| UGT1A1 [ Felis catus ] 98.6 1e-19 gi|31324698|gb|AAP48597.1| UGT1A9 [ Mus musculus ] 98.6 1e-19 gi|31657196|gb|AAH53576.1| UGT1A10 protein [ Homo sapiens ] 98.6 1e-19

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103 Figure 5-18. Results of NCBI conserved domain search for I35R_Cp The protein sequences obtained from liver and intestine were aligned, showing a strongly conserved sequence for all three at residues 371-391 (relative to liv/intUGTn, Figure 5-19), which corresponds to the UDPGA binding site.

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104 Scores SeqA Name Len(aa) SeqB Name Len(aa) Score ========================= ================== ============ 1 livUGTp 627 2 I35R_Cp 133 60 ========================= ================== ============ livUGTp GMLRARVPGLFFSHSSSFFFSSKICFL-T-LETFKLKMPRLLAALCLQIYLCSFLGPVEG 58 I35R_Cp -----------------------------------------------------------livUGTp GKVLVMPVDGSHWLSMKILVEELSRRGHEMVVLVPETSVLIHGSDAYVARSFKVPYTKAE 118 I35R_Cp -----------------------------------------------------------livUGTp LDESMNKLKEGITKAPRISDLLENIIGLLSFTNMQVKGCEALLYNEPLMQNLREEHFDLM 178 I35R_Cp -----------------------------------------------------------livUGTp LTDPFLPCGPIIAEAFSLPAVYFLRGLPCGLDLEAAQCPSPPSYVPRFFTGNTDVMTFSQ 238 I35R_Cp -----------------------------------------------------------livUGTp RVKNVLMTGFESILCKIFFSSFDELTSRYLKKDVTFRDVLGHAAIWLYRYDFTFEYPRPV 298 I35R_Cp -----------------------------------------------------------livUGTp MPNAVRIGGINCAKKNPLPADLEEFVDGSGDHGFIVFTLGSFVSELPEFKAREFFEAFRQ 358 I35R_Cp ----------------------------PANTTWILWSKTHTEGH----HTVPVLNSVKI 28 ..: :*::: .. :: .:::.: livUGTp IPQRVLWRYTGVIPKDIPENVKVMKWLPQNDLLAHPKAKVFITHGGAHGIYEGICNGVPM 417 I35R_Cp -----I-KIRGNI--MVHNNNFLFSFLSSPC-KAHPKAKVFITHGGTHGIYEGICNGVPM 79 : : * * : :* :: :*.. *************:************* ----------UDPGA binding site --livUGTp VMIPLFGDQVDNVLRMVLRGVAESLTMFDLTSEQLLGALRKVLNNKRYKEKITQLSLIHK 477 I35R_Cp VMIPLFGDQVDNVLRMVLREVAESLTMFDLTSEQLLGALRKVLNNER-KK---------128 :*:**************** *************************:* *: ---------livUGTp DRPIEPLDLAVFWTEFVMRHGSAEHLRPAAHHLNWVQYHSLDVIAFLLLVLSTVVFIAVK 537 I35R_Cp -----------------------------------------------------------TM region-livUGTp TCALCFRKCFRRAQKSKKE-NGQ-MIRNGFGAVFN-RRWFIGVMSYCENLK-L-CSHHHV 592 I35R_Cp -----------------------------------------------------------livUGTp QFNIQGCQQLWFSHCSYGCYDVTKKKK 619 I35R_Cp --------------------------Figure 5-19. Alignment of predicted prot ein sequences from cloned catfish UGTs. Regions of interest and the starting a nd ending residue of the mature product are highlighted. Cloning of entire UGT gene The liver UGT gene was successfully amp lified and cloned, using sets of primers that were complementary to both the unt ranslated regions (UTRs) upstream and downstream of the gene, as well as primers that annealed to the exac t start and end of the translated portion of the gene (Figure 5-20). The same PCR conditions were used in an

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105 attempt to amplify a product from intestinal cDNA, but th is was successful only for the primers that annealed to the unt ranslated sequences (Figure 5-21). A B 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Figure 5-20. Cloning of livUGTn. A. Amplification of UGT gene with (lan es 1-6) and without UTR (lanes 7-9), using various primer combinations and annealing temperatures. B. UTR insert (lanes 1-3) and UGT insert (lanes 4-6) cloned into p-GEM T-Easy vector, total size ~ 4500bp. 1kb ExactGene DNA ladder s hown in first lane (0) of both pictures. A B 0 1 2 3 4 0 1 2 3 4 Figure 5-21. Cloning of intUGTn. A. Amplification of UGT gene with (lan e 3) and without UTR (lane 4); lanes 1 and 2 show unsuccessful amp lification with degenerate primers. B. UTR insert (lanes 1-4) cloned into p-GEM T-Easy vector, total size ~ 4500bp. 1kb ExactGene DNA ladder shown in first lane (0) of both pictures. 1500 1500

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106 The sequence of the DNA represented by th ese clones was compared to the UGT sequence deduced from the partial sequen ces of the various overlapping clones and amplicons (Table 5-13). There were mini mal variations (99% homology), with the exception of a triple base change from AAA to TTT in all the clones at position 648. Table 5-13. Results of ClustalW multiple se quence alignment analysis of the cloned UGTs and the original livUGTn SeqA Name Len(nt) SeqB Name Len(nt) Score ================== ================== ================= 1 livUGTn 1566 2 UTR1 1782 99 1 livUGTn 1566 3 UTR2 1799 99 1 livUGTn 1566 4 UTR3 1798 99 2 UTR1 1782 3 UTR2 1799 99 2 UTR1 1782 4 UTR3 1798 99 3 UTR2 1799 4 UTR3 1798 99 ================== ================== ================= SeqA Name Len(nt) SeqB Name Len(nt) Score ================== ================== ================= 1 UGT1 1569 2 UGT2 1569 99 1 UGT1 1569 3 UGT3 1569 99 1 UGT1 1569 4 livUGTn 1566 99 2 UGT2 1569 3 UGT3 1569 99 2 UGT2 1569 4 livUGTn 1566 99 3 UGT3 1569 4 livUGTn 1566 99 ================== ================== ================= For full-sequences of these full-length UGT clones, refer to Appendix B.

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107 Discussion Multiplicity of UGT isoforms. At least two different UGT isoforms were identified from channel catfish liver (livUGT n) and intestine. One isoform (liv/intUGTn) was sequenced in its entirety and was clone d from both liver and intestine. Another isoform (I35R_C) was sequenced from intestine; amplification of this partial sequence towards its 5 and 3 -ends was unsuccessful. However, the partial sequence obtained, particularly a 45-bp st retch at the known 5 -end of this sequence (w hich includes part of the UDPGA binding site) was significantly di fferent from the se quence of the other isoform when both sequences were aligned. The liv/intUGTn sequence appeared to be analogous to mammalian UGT1A1, or bilirubin UGT, while blastp searches for the predicted partial protei n sequence of I35R_C resulted in better matches with the highe r-numbered UGT isoforms such as UGT1A4, UGT1A7, UGT1A6. Of course, one cannot c onclude anything fu rther since these sequences are only partial, lack ing the substrate-binding site which is responsible for the distinct specificity of the UGT isoform. The presence of different UGT isoforms in catfish liver and in testine are probably one of the reasons for the di sparate glucuronidation kinetics observed in these organs with substrates such as 3-hydroxybenzo[a]pyr ene and polychlorinated biphenylols. Characteristics of the predicted protein for livUGTn. As seen from Figure 5-13, the UGT isoform obtained from catfish show s several strongly cons erved regions with mammals, even though 350 million years of evolution separate the two phyla. These indicate amino acid residues that are im portant for the function of the protein. By drawing an analogy with mammalian UGTs, which have been extensively studied, several interesting f eatures regarding the catfish UGT sequence were noted. Two

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108 regions were clearly distingui shed in the C-terminal doma in, that is the UDPGA binding site at residues 347-394 (Mackenzie et al., 1997), and the transmembrane domain at residues 499-517. The cytoplasmic tail ends w ith the double lysine retrieval motif (KSKKE); in mammals this is –KSK TH (Radominska-Pandya et al., 2005) The UDPGA-binding region is highly c onserved. The two basic residues K347 and K359 may form Schiff-base adducts with the uridinyl moiety as part of the process of transfer of glucuronic aci d to the substrate (Radomin ska-Pandya et al., 1999). The histidine residue at 357 in teracts with UDPGA while 366His is required for catalysis in UGT1A6 (Battaglia et al., 1994) . Further downstream, the 441-504 region is also highly conserved; it contains a 442Lys and 443Asp that help to maintain the active conformation of the enzyme (Iwano et al., 1999), as well as a 481His that is one of 6 histidine residues important for the catalytic activity of human UGT1A6 (Battaglia et al., 1994). The most variable amino acids for mamma lian UGT sequences are located between amino acid residues 55-180. We observed, howev er, some strongly conserved regions even in this region, as well as further downstr eam (Table 5-14). One region of interest in UGT1A proteins is where a c onserved hydrophobic region is t hought to be the site of a buried -helix containing an essential Phe that is critical for bilir ubin glucuronidation (Ciotti et al., 1998). In the catfish UGT, there is an equivalent hydrophobic region VYF166L (Fig 5-16). In mammalian steroid UGT s (UGT2B family), this region is disrupted by a Ser, thus providing further evid ence that the catfish UGT is more likely a homolog of the mammalian UGT1 family.

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109 Table 5-14. Conserved consecutive residues observed in catfish liver and mammalian UGTs (sequences shown in Figure 5-13). X indicates unconserved residue Residue Start Sequence Importance Reference 32 GS H WLSM Contains His which is cr itical for Battaglia et al., 1994 activity 49 RG H Conserved hydrophobic regi on; Battaglia et al., 1994 His required for optimal enzyme Senay et al., 1997 activity but not involved in catalysis 54 VVLVP Important for enzyme function Senay et al., 1997 144 LTDPF Unknown 199 M T F X QRVKN X L Contains a phosphokinase site (T) Basu et al., 2005 249 WL X R X DF Unknown 263 M P N X VI GG INC Pro and Gly important fo r UGT1A1 Ciotti et al., 1995 activity and secondary structure 299 VFS/TLGS X VSEI/LP Unknown 324 IPQ X VLWRYTG Unknown 424 VL/INNK X YKE Unknown A high degree of conservation (77%) was present for proline residues between catfish and the mammalian UGTs. The unique structure of this cyclic amino acid permits twists and kinks in the proteinÂ’s tertiary stru cture. This indicates that overall, the threedimensional structure of cat fish liver UGTs is similar to its mammalian homologs. On the other hand, there were some important differences. The Ile211 residue which is essential for activity in human UGT1A10 (Martineau et al., 2004), was replaced by a Leu in channel catfish (as in human UGT1A1, UGT1A3, UGT1A4, UGT1A6). The phenylalanine residues at positions 90 and 93 wh ich have shown to be important for the catalytic activity of UGT1A10 towards phenols such as para-nitrophenol and 4methylumbelliferone (Xiong et al., 2006), were absent in the catfish sequence as well as other fish sequences (Figure 5-22). Furthe r upstream, the strongly conserved binding motif Y73/72XX TK X YPVP that has been show n to be involved in the binding of phenols in

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110 the case of mammalian UGT1A1 and UGT1A6 respectively (Senay et al., 1999) was discernible in the fish sequences, including catfish. The substitution of Ala for Thr at position 72 in catfish may provide additional evidence that the cloned UGT is not an analog of mammalian UGT1A7 and UGT1A10, si nce other studies have shown that mutation of this highly conserved residue led to a total inactivation of these isozymes, possibly due to the alteration of phosphorylation state of the enzyme (Basu et al., 2005). Â…Â…Â…Â…Â…Â…Â…Â… Â…Â…Â…Â… D.rerio_AAI09405.1 TSILIKKSGKYSTKTYPVSFTHDDLAENLKEIQNSALEK--APKLTDIVV 109 D.rerio_AAH75892.1 TSILIKKSGKYSTKTYPVSFTHDDLAENLKEIQNSALEK--APKLTDIVV 107 D.rerio_NP_998587.1 TSILIGKSGNFTTKSFRVPYSFDELNAHVDHIRKTAIEK--APRFIDIVG 110 D.rerio_AAH93347.1 ASLSMGPSEKTTTLTYPVNYTKAELHMVLEGNLTEILSTDFSTEVSKFFV 104 T.nigroviridis_CAG04937.1 TSLLIKSSENYRTEIYQVPYSKEDLGGSFQALKDGLFLK--PPSMADLFV 110 T.nigroviridis_CAF91810.1 TSLLIKSSENYRTEIYQVPYSKEDLDGSFQALKDGLFLK--PPSMADLFV 110 E.coioides_AAW29020.1 SSLLIHGSESYKTEIYQVSYTKAELDGKFAELQTGVSLK--PPAITDLFI 62 P.platessa_CAB51368.1 SSLFMHQSEDYKTEVYPVSFTTEEMDATHKQLKDGLFLK--QPDWTEYYV 110 P.platessa_CAB51369.2 SSLFMHQSEDYKTEVYPVSFTTEEMDATHKQLKDGLFLK--QPDWTEYYV 110 P.platessa_CAA52214.1 SSLFMHQSEDYETEVYPVSFTTEEMDATHKQLKDGLFLK--QPDWTEYYV 52 P.yokohamae_BAC87829.1 SSLFMHQSEDYKTEVYPVSFTMEEMDAVHKQLKDGLFLK--QPDWTEYYV 110 I.punctatus_livUGTp TSVLIHGSDAYVARSFKVPYTKAELDESMNKLKEGIT-K--APRISDLLE 141 D.rerio_XP_687385.1 ISMRLGPGKHYITKKFPVKYDQKLFNEVLTEHVHEVTNPG-HSRLKTVTS 398 D.rerio_XP_686831.1 VSVLLGPGKHYVTRTFPVLYGKQQLDELQARNAQVMESKQ-LPLMEKIST 126 D.rerio_AAI00056.1 KNILIQSSELFRTETFPVKISKEQLSKSLKGFQQGVFTR--SPALMDVFV 113 .: : . : : * : Figure 5-22. Multiple sequence alignment for fish sequences homologous to catfish UGT isolated from liver and intestine, show ing regions where substrate binding of phenols is thought to occur for mammalian UGT1A isozymes. Upstream highlighted eight residue-l ong sequence represents putative phenol binding site for UGT1A6 and UGT 1A 1; downstream highlighted four residue-long sequence represents the phe nol binding site for UGT1A10. A potential phosphorylation site analogous to that observed for human UGT1A7 (Basu et al., 2005) may al so be present at Thr200; however other sites which have been shown to be phosphorylated, notably Ser432, are absent in the cloned catfish UGT (but present in the fish sequences listed in Figure 5-10). Limitations 3 -truncated UGT sequences. These were obtained for both liver and intestine. For example, the 3 RACE performed in order to ex tend the UGT liver sequence resulted

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111 600in three bands (Figure 5-23). The 700bp and 300bp bands were cloned and sequenced and were found to be identical except fo r the fact that the 300bp product had a polyadenylated tail 400 bases upstream (in an adenine-rich region) of the polyadenylated tail belonging to the la rger product. Cloning of th e 1,200bp product was unsuccessful. Figure 5-23. Results of 3 RACE performed on liver, showing multiple products obtained. In addition, it was also observed that further amplifying a PCR product obtained via RACE in order to increase the yield of this product, ofte n resulted in the formation of smaller products. For example, the 3 RACE performed in order to extend I4_degenerate resulted in two products which showed up as an intense 300bp band and a faint 700bp band (which was the expected product size) (F igure 5-24A). The gel piece containing the larger amplicon was purified and then subject ed to an additional round of PCR (Figure 524B). The result were three bands: an expe cted one at 700bp (whi ch was sequenced and corresponded to the sequence containing a full-length 3 end (I4_3R)), and two smaller bands approximately 300 and 200bp in size. This could mean that there is some form of 1500 300

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112 degradation. Lacking any ot her explanation, it was tentat ively concluded that these truncated UGT sequences were artifacts of the PCR reaction. A B Figure 5-24. 3 RACE for I4. A. Inner PCR, B. Additional round of PC R (PCR reaction loaded in duplicate) Quality of RNA. The fact that only two UGT isof orms were identified indicated some problem with the methodology. Catfish, lik e the phylogenetically related zebrafish, was expected to have multiple dissimilar UGT isoforms, particularly in liver. The fact that RACE failed to amplify the partial DNA sequences on several occasions led to the postulation that the RT reaction had been performed at a temperature that was too low (42°C, even though this is within the norma l operating temperatures for the MMLV-RT enzyme), resulting in mRNA that was not fold ed correctly. In addi tion, it was noted that the RACE procedure omitted the heating step for 3 min at 70-85°C, prior to the actual reverse transcription, which helps to unfold th e RNA. However, the heating step had been 700 700

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113 used to generate cDNA in the initial PCR using the degenerate primers, which resulted in only two successful amplifications for the liver and one for the intestine (Figure 5-4). The RT reaction was reperformed at 50°C, but this did not seem to increase the number of amplicons. Thus, it was unlikely that the proble m was in the reverse transcription step. When the degenerate primers were rerun for cDNA originating from the intestine of another channel catfish (AT17), and cDNA from largemouth bass ( Micropterus salmoides ) liver, a larger number of amplicons (o f expected sizes) were generated (Figure 5-25). In addition, the bands representing these various amp licons were more intense. The UGT cloned from catfish intestine derive d from AT17 and not AT45. These findings indicate that the quality of the original RNA prepared from catfish AT45 intestine (and possibly liver) was not satisfact ory. One reason for the difference between the RNA of both intestinal samples could have been due to the fact that while the intestinal RNA that was used originated from mucosa that had been scraped off the smooth muscle wall of the intestine, the AT17 intestinal RNA was derived from a tissue sample that was processed without separation of the mucosa from the underlying muscle. It is possible that the process of scraping the mucosa, even though this was done on ice and only lasted for a few minutes, caused the degradation of a significant proporti on of the UGT mRNA population, resulting in the gene ration of a limited cDNA libr ary. This may occur due to stimulation of the secretion of proteases, nucleases, and other hydrolytic enzymes. On one previous occasion, the proce ss of scraping the catfish inte stinal mucosa resulted in poor quality cDNA for a CYP450 cloni ng study. Subsequently, the CYP450 was successfully cloned from a sample that wa s derived from RNA originating from a transverse section of the intestine (D r.David Barber, personal communication).

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114 A. B. C. 0 1 2 3 4 5 6 7 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 Figure 5-25. PCR amplification of UGT using degenerate primers. In all cases, the same seven sets of primers were used under similar PCR conditions. cDNA templates were as follows: A, channel catfish AT45 intestine; B, channel catfish AT17 intestine; C, largemouth bass liver (lane 8 is similar to lane 6, but the PCR was r un at a higher annealing temperature). Lane 0 represents 100kb ladder for (A) a nd 1kb ladder for (B) and (C). Conclusions and recommendations One full length UGT from catfish liver, t ogether with an identical UGT from catfish intestine, was successfully cloned. A partial sequence of another UGT from catfish intestine was also cloned. By homol ogy with mammalian UGTs, the full-length catfish UGT clone appeared to be analogous to UGT1A1 or UGT1A6. Expressing this gene into suitable cells (e.g. V79 or HEK ce lls) and characterizing the resulting protein should provide us with further information on glucuronidation in the catfish. Performing enzyme assays with UGT-selective probes, such as bilirubin (UGT1A1) and serotonin (UGT1A6) (Patten et al., 2001; Krishnaswami et al., 2003), would assist in such studies. As Table 5-11 shows, there ar e several potential antigenic sites on the predicted protein sequence that may be exploited to design specific anti-UGT antibodies which would

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115 recognize such epitopes, in orde r to study the different levels of UGT protein in various tissues or in response to envir onmental stressors such as xenobi otic inducers or inhibitors. The availability of such isoform-selective antibodies is lacking, even for human UGTs (Miners et al., 2006). Additional samples of catfish liver and intestinal RNA s hould be obtained, ensuring that the RNA is of optimal quality. After a numb er of isoforms have been isolated, realtime PCR could be performed using cDNA from catfish tissues such as brain, kidney, gills and skin to investigate the distribu tion of different UGT mRNAs in the channel catfish. Other studies can be performed on ge nomic DNA, in order to understand the gene locus for catfish UGT and whether differentia l splicing does indeed occur as in mammals. This would also be an opportunity to identi fy HNF-1 binding sites which reside within proximal upstream regulatory regions of human UGT genes (Gardner-Stephen et al., 2005) as well as the distal enhancer module which is the site of binding of nuclear receptors such as the glucocorticoid and the pregnane X receptor in mammalian UGTs (Sugatani et al., 2005).

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116 CHAPTER 6 DETERMINATION OF PHYSIOLOGICA L UDPGA CONCENTRATIONS IN CHANNEL CATFISH LIVER AND INTESTINE UDP-Glucuronic Acid (UDPGA) When trying to determine Km (or S50) and Vmax values for the substrate of interest in a bisubstrate system such as glucuroni dation, one keeps the c oncentrations of cosubstrate constant. This is done since, in th eory, by using different concentrations of cosubstrate one can get an infinite variety of values ( apparent values ) for the kinetic parameters. Thus, in order to obtain true values via the Michaelis-Menten equation, one has to determine kinetic parameters at satura ting concentrations of co-substrate, assuming that these excess concentrations do not have any other effect on UGTs. Thus, for example, saturating concentrations of 0.2 mM UDPGA were used in our catfish intestine glucuronidation experi ments (Chapter 4). However, the use of excess concentra tions of UDPGA present one with two problems. Are UDPGA concentrations saturating in vivo ? How is the enzymatic efficiency affected, in view of the affinity (shown by Km or S50) of UGT(s) for UDPGA? If the physiological UDPGA concen trations are lower than th e excess concentration used in vitro one may expect to observe a difference in kinetic parameters. While some studies have measured the physiological UDPGA level in tissues (mainly liver), most of this work has been limited to mammalian species such as humans and rats (Table 6-1). The rate of glucuronidation of 3-OH-B[a ]P was also found to be dependent on the endogenous level of UDPGA by Singh and co-wor kers (1986). This concept of UDPGA

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117 supply as a rate-lim iting factor has been observed in the glucuronidation of 7hydroxycoumarin (Conway et al., 1988) and acet aminophen given together with retinol (which depletes UDPGA stores ) (Bray and Rosengren 2001). Table 6-1. UDPGA concentrations ( M) in liver and intes tine of various species Species Liver Small intestine Reference Human 279, 301 119 19.3 4.5 Cappiello et al., 1991, 2000 Human fetus 59.4 11.3 Cappiello et al., 2000 Rat 200-500 121 5 70 crypt cells 200 villus cells Hjelle et al., 1985, Goon and Klaassen 1992, Yamamura et al., 2000, Dills et al., 1987D, Pang et al., 1981, Hjelle 1986, Zhivkov et al., 1975 D, Dubey and Singh 1988 Mouse 201 17.6 Bray and Rosengren 2001 Guinea-pig 413 3 322 – 580 79 6 stomach 36 3 colon Zhivkov et al., 1975 D, Singh et al., 1986 Pig Cat Rabbit 292 24 153 182 20 Zhivkov et al., 1975 D Chicken Turkey Pigeon 51 6 124 26 78 13 Ibid Frog Newt 81 11 73 5 Ibid Trout Carp 116 8 21 3 Ibid D refers to a direct method of UDPGA meas urement. All other references relied on indirect methods of UDPGA de termination (see discussion). Biphasic UDPGA kinetics have been dem onstrated for 1-naphthol, morphine, 4methylumbelliferone, and 3-hydroxybenzo[a]pyrene (Miners et al., 1988a,b; Tsoutsikos et al., 2004; Chapter 3). Whether this was due to the presence of multiple enzymes or an

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118 allosteric effect by UDPGA on substrate bi nding (as proposed by Ethell and Wrighton 2004) is not known. Does the low-affinity co mponent of the bipha sic glucuronidation exist in vivo , or is it an in vitro artifact of the exce ss UDPGA concentrations? In summary, the implication here is that the UGT activity obtained using saturating concentrations of UDPGA that are in excess of physiological concentrations in a certain tissue belonging to a specific sp ecies can render extrapolating in vitro data to in vivo situations potentially useless. One can overe stimate the efficacy of glucuronidation of a xenobiotic because the maximal rate determ ined by conventional kinetic experiments may be greater than the maximal rate in vivo . In addition, compounds which are glucuronidated at rates rangi ng from the low nmol/min per mg to <1 pmol/min per mg may all be regarded as substrates in the presence of high concentrations of UDPGA (Miners et al., 2006). Objective To develop a reproducible method for the determination of UDP GA concentrations in channel catfish liver and intestine. Method Development Previous attempts to separate UDPGA by HPLC utilizing a C18 or a C4 column gave rise to results which were not reproduc ible. It was decided that, since UDPGA is acidic at physiological p H, an ion-exchange column would be used in order to separate this compound from other components of biolog ical tissue that absorb at 260 nm (mainly nucleotides and nucleotide sugars).

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119 Sample Digestion The effect of boiling on UDPGA stability was investigated both with regards to heating time as well as the chemical nature of the solution in which the UDPGA was dissolved in. Boiling in water for 3 minutes resulted in a 12.7% loss of UDPGA (present as standard solution), comp ared to boiling in 0.25M H2PO4 for the same amount of time (7.7% loss). A final boiling time of 1 min was chosen since measurement of the temperature inside the glass tube showed that , within 15 seconds, the temperature of the water within the tube rose to 98°C when th e tube was plunged into briskly boiling water. In addition, UDPGA loss when boiling in buffe r for this period of time was minimal (Figure 6-1), and did not lead to appreciab le decomposition of UD PGA to UDP and UMP (Bedford et al., 2003; Figure 6-2). Boiling of the liver sample for 1 minute in 0.30 M buffer, p H 4.3 resulted in less sample loss than boiling for 1 min or 3 min in 0.25 M buffer, p H 3.4 (Figure 6-3). Thus the final samp le treatment conditions chosen were boiling for 1 minute in 0.30 M H2PO4 in H2O, p H 4.3. While liver samples were boiled in buffer as a 1 in 5 dilution ( 0.1 g liver with 0.4 mL buffer), intestinal samples were boiled as 2 in 5 (0.2 g with 0.3 mL), since UDPGA concentrations in intestine were expected to be significantly less than in liver.

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120 0.0 2.5 5.0 7.5 10.0 12.5 0 10 20time (min)%UDPGA lost Figure 6-1. Heat-induced degradation of UDPGA (boiling in 0.25 M H2PO4 buffer) Figure 6-2. Decomposition of UDPGA to UDP and UMP after boiling in 0.25 M H2PO4 buffer for 10 minutes 0:00 5:00 10:00 15:00 20:00 25:00 30:00 min 0 10 20 mVUMP ? UDP UDPGA

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121 A B Figure 6-3. Effect of boiling li ver tissue for 1 minute in two different concentrations of buffer. A, 0.25 M H2PO4, p H 3.4; B, 0.30 M H2PO4, p H 4.3 HPLC A mobile phase consisting of 0.3 M NH4H2PO4 in water ( p H 3.1) was initially tried. UDPGA standards eluted at 16 min. However, this elution time was found to be 0:00 5:00 10:00 15:00 20:00 25:00 30:00 35:00 40:00 min 0 10 20 30 40 mV 1 min boiling in 0.30M buffer, pH 4.3UDPGA 0:00 5:00 10:00 15:00 20:00 25:00 30:00 35:00 40:00 min 0 10 20 30 mV 1 min boiling in 0.25M bufferUDPGA

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122 unsatisfactory when liver samples were test ed, due to the proximity of various other peaks which led to a drifting baseline and inaccurate estimations of peak area. A decrease in buffer concentration to 0.25 M, with p H at 3.4, resulted in an increased retention time of 26 min. This is to be expected si nce the decreased concentration of H2PO4 ions results in decreased competition with the UDPGAions for the column bound NH4 +. The net result was a well resolved peak corresponding to liver and intestinal UDPGA (Figures 64 and 6-5). The decreased retention time for UDPGA in the case of intestine may be due to differences in the sample matrix arising from the smaller diluti on used in the initial homogenization step (p.120). Figure 6-4. HPLC chromatogram for catfish AT 17 liver. Center refers to region of liver from which the sample was taken. 0:00 5:00 10:00 15:00 20:00 25:00 30:00 35:00 40:00 min 0 10 20 30 40 50 mV AT17 liver (center)UDPGA

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123 Figure 6-5. HPLC chromatogram for catfish AT18 intestine. Rep 2 refers to second sample taken from AT18 intestine. Final Method Liver tissue, 0.1 g, or 0.2 g intestinal muco sa were placed at the bottom of a small thick glass homogenizing tube (Thomas AA717 ). Using a rough tipped pestle attached to an electric drill, the tissue was homogenized for 10 seconds w ith 0.4 or 0.3 mL (liver or intestine respectively) of 0.3 M NH4H2PO4. The tube in the homogenate was then placed in a briskly boiling water bath for 1 minute af ter which it was removed and placed on ice. The boiled mixture was briefly rehomogeni zed. The tubes were centrifuged, and the supernatant (still containing suspended matter) was transferred into a 1.5 mL microfuge tube and recentrifuged for 15 minutes at 16,000 g. The supernatant was then filtered by spin-centrifugation (using 0.45 M Spin-filters (Costar, Co rning Inc., NY)) at 16,000g for 5 minutes. A sample, 50 L, was analyzed by HPLC (Model 2300 pump (ISCO, Lincoln, NE) with Dynamax UV absorban ce detector (Raini n, Woburn, MA)). 0:00 5:00 10:00 15:00 20:00 25:00 30:00 35:00 40:00 min 0 10 20 30 40 50 mV AT18 INT REP2UDPGA

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124 HPLC conditions involved an is ocratic system passing 0.25M NH4H2PO4 (HPLC grade) in water ( p H 3.4) at 1mL/min through a Zorbax SAX column (4.6mm i.d. x 250 mm, 5 M) with UV detection at 260 nm. The el ution times of so me physiologically important chemicals, including UDPGA, are given in Table 6-2. Table 6-2. Elution times of certain physiol ogical substances (standards dissolved in mobile phase1) using the anion-exchange HPLC conditions described above. Compound Approximate elution time (min) __________________________________________________ UDP-glucuronic acid 23 UDP-glucose 7 UDP 18 UDP-galacturonic acid 21 PAPS1 45 ___________________________________________________ 1 The PAPS (3 -phosphoadenosine-5 -phosphosulfate, co-subs trate for sulfonate conjugation) standard was dissol ved in water in view of uncer tainties regarding its heat lability and/or acid stability. UDP-galacturonic acid, an epimer of UD PGA, could be resolved from UDPGA using this method (Figure 6-6). The useful ness of anion-exchange chromatography in separating these structurally related nucleotide sugars has been demonstrated elsewhere (Liljebjelke et al., 1995; Sc hlüpmann et al., 1994).

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125 Figure 6-6. HPLC chromatogram of UDP, UDP-galacturonic acid (UDPGTA), and UDPGA standards. Results Several catfish liver and whole intestines were analyzed for UDPGA content using this method. The livers had been put in plastic tubes (with no buffer) immediately following sacrifice, and stored at -80°C. A paired t-test reveal ed that mean concentrations in liver were significantly higher than in intestine ( p =0.008, Table 6-3 and Figure 6-7). Replicates showed less than 25% SD except fo r two fish (AT17 and AT18). Interestingly, the intestinal UDPGA values for one fish (AT17) were comparable to the hepatic UDPGA values of another fish (GS39). 0:00 5:00 10:00 15:00 20:00 25:00 30:00 min 0 10 20 30 40 50 mVUDP UDPGTA UDPGA

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126 Table 6-3. UDPGA concentrations in M (duplicates for individual fish), in catfish liver and intestine. Fish Liver Intestine --------------------------------------GS38 332, 326 ND1 GS39 252, 242 ND1 AT17 368, 397 235, 281 AT18 287, 355 174, 173 AT19 368, 376 87, 116 AT20 437, 426 87, 68 AT45 429, 458 59,71 Mean 361 ± 682 135 ± 812 -------------------------------------1 ND, not performed due to unavailability of tissue 2 Standard deviation of the mean AT17 AT18 AT19 AT20 AT45 0 100 200 300 400 500Liver Intestine Fish[UDPGA] (M) Figure 6-7. Comparison of hepatic and intes tinal [UDPGA] in 4 individual channel catfish.

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127 Discussion Two major strategies have been used to study UDPGA concentrations in tissues. Indirect determination of UDPGA concentrations is ba sed on the normally linear relationship between glucuronide form ation and UDPGA concentration. The determination of glucuronide formation, whethe r via radiochemical de tection (Schiller et al., 1982; Watkins and Klaase n, 1982; Hjelle et al., 1985; Cappiello et al., 1991), fluorometry (Singh et al., 1986) or revers e-phase HPLC (Yamamura et al., 2000), can then be used to down-extrapolate the UDPGA level via a standard curve. Since this method assumes that the lin ear relationship holds at low UDPGA physiological concentrations, determination of this co-substr ate in tissues with lower levels (such as intestine) may be more subj ect to inaccuracies. Reverse phase HPLC has been used to di rectly determine UDPGA concentrations in liver cell extracts (Aw and Jones, 1978; D ills et al., 1987; Alary et al., 1992) and whole tissue (Adachi et al., 1991; Suto et al., 2002 ). Imamura and co-workers (2003) used a reverse-phase system in order to determine both UDPGA and PAPS in cultured rat hepatocytes. While direct determination of UDPGA by HPLC is most desirable, the use of a C18 reverse-phase column was unsucce ssful, due to the interference of other substances co-eluting with the UDPGA peak, as well as a drifting baseline. The use of an anion-exchange HPLC column dramatical ly improved resoluti on, sensitivity and reproducibility. Sub-micromolar concentra tions of UDPGA standard, dissolved in ammonium phosphate buffer, could be detected. Catfish liver UDPGA concentrations were similar to those previously reported for mammals such as humans, rats, and guinea pi gs (Table 6-1). The results reported by Zhivkov and co-workers (1975) for other mamma lian species, birds, amphibians and fish

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128 are much lower than the catfish UDPGA leve ls measured with this method. Intestinal UDPGA concentrations in the catfish reported here are the first, to our knowledge, ever to have been reported for the intestine of an y piscine species. These concentrations were in the same range as that reported for rats, but higher than humans and lower than guinea pigs. While some of these differences are sp ecies-related, another important contributor to the discrepancy is the different analytical techniques, some of which are inherently limited by an indirect measurement of UDPGA. Another possible source of variation may have been the dietary status of the indi vidual animal, since UDPGA concentrations are decreased by fasting (Reinke et al., 1981). Th e only values measured in fish are those measured by Zhivkov and co-workers (1975), who homogenized liver tissue in perchloric acid in order to solubilize the nucleotides. This may have led to the lower values observed in trout and carp liver (Table 6-1) relative to catfish liver, since the rate of hydrolysis of UDPGA to UDP has been shown to be proportional to hydrogen ion concentration (Bedford et al., 2003). Hepatic UDPGA concentrations were in the range of 329-444 M. The UDPGA Km values obtained for the he patic glucuronidation were 247 M and 697 M for 4 OHCB-72 and 4 -OHCB-35 respectively (Table 4-1). This means that, in vivo , at the saturating concentration of substrate used in the assay, and assuming that this UDPGA concentration range is typical of catfish populations, hepatic glucuronidation proceeds at a suboptimal rate for both 4 -OHCB-72 and 4 -OHCB-35. Of course, one must remember that the substrate concentrations used in the assay were not representative of environmental concentrations, and thus at smaller, more realistic OH-PCB levels, the UDPGA concentration is pr obably sufficient to effi ciently glucuronidate 4 -OHCB-35.

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129 Intestinal UDPGA concentrations appeared to have a larger range than in liver (65258 M). The decreased UDPGA concentrations (re lative to liver) are reflected in the decreased UDPGA Km of 27 M reported for 4 -OHCB-69 (Table 4-1). The UGT isoforms in the intestine responsible for OH-PCB glucuronidation ope rate in a cellular environment with decreased UDPGA concentrati ons and thus appear to work optimally at lower concentrations of co-substrate. The physiological hepatic levels of UDPGA are about 4 times lower than the 1.5 mM co-substrate concentrations utilized in the OH-PCB glucuronidation study in catfish (Chapter 4). In contrast, the physiological intestinal levels of UDPGA are around the same concentration as the amount used in the UGT assay (200 M). This means that the Vmax reported for hepatic OH-PCB glucuronida tion in Chapter 4, probably represent overestimates that would not be achievable in vivo . Conclusions and Recommendations A method to directly determine UDP GA in tissue by anion-exchange chromatography was developed and used to study UDPGA concentrations in channel catfish liver and intestine. The method wa s sensitive, reproducible and displayed good resolution for UDPGA. The technique may be adapted to study other nucleotide sugars. The hepatic UDPGA levels determined by this technique were similar to those in other mammalian species and higher than two other fish species. This was the first time intestinal UDPGA concentrations in any piscine species were determined; the values were similar to rat, but significantly hi gher than in human small intestine. Future studies should determine the UDPGA concentrations in a greater number of catfish, as well as in different tissues , including determination of the UDPGA

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130 concentrations in the proximal a nd distal parts of the intestine. The effect of diet on the concentrations of this co-subs trate in the experimental anim als should also be taken into consideration. These studies should be perf ormed in conjunction with experiments on glucuronidation kinetics, so as to better extrapolate in vitro findings to the in vivo situation.

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131 APPENDIX A SEQUENCES OF UGT PARTIAL CLONES AND AMPLICONS The information given in parentheses after the title of each sequence provides data on the experiment which generated the sequen ce, together with photographic evidence. This includes Lab Book number and page, as well as the exact lane which shows the agarose gel-purified DNA. A. 5' 3' sequences of partial length clones for liver UGT i. SEQUENCES OBTAINED BY DEGENERATE PRIMERS >L1_DEGENERATE (LB XI, p. 86, lane 1) GAGTTTGTGG ATGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA TGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAA CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GCTGATGTTT CCTCTGTTT >L2_DEGENERATE (LB XI, p. 86, lane 2) GAGTTTGTGG ATGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA TGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAA CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GCTGATGTTC CCTCTGTTT >L3_DEGENERATE (LB XI, p. 86, lane 3) GAGTTTGTGA AAGGCTCTGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA CGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAG CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GCTGATGTTC CCGCTGTTT

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132 >L4_DEGENERATE (LB XI, p. 86, lane 4) GAGTTTGTCG AAGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA CGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGTG CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GTTGATGTTC CCGCTGTTT >L5_DEGENERATE (LB XI, p. 86, lane 5) GAGTTTGTGA AAGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA CGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAG CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GTTGATGTTC CCGCTGTTT >L6_DEGENERATE (LB XI, p. 86, lane 6) GAGTTTGTGA ATGGCTCTGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA TGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAA CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GGTGATGATC CCGCTGTTCG GAGATCAGGT AGACAACGTT CTACGCATGG TGCTGCGTGA AGTCGCAGAG AGCCTGACCA TGTTCGACCT GACCTCAGAG CAACTGCTGG GGGCACTCAG GAAAGTCCTC AACAACAAGC GCTACAAAGA GAAGATAACA CAGCTGTCTT TGATCCATAA AGACCGTCCG ATCGAGCCGC TGGACTTGGC CGTGTTCTGG ACCGAGTTTG TGATGAGACA CGGAAGTGCC GAGCACCTGA GACCGGCCGC TCACCACCTG AACTGGATTC AGTACCAC >L7_DEGENERATE (LB XI, p. 86, lane 7) GAGTTTGTGG AAGGCTCTGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA CGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAG CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GGTGATGATC CCGCTGTTCG GAGATCAGGT AGACAACGTT CTACGCATGG TGCTGCGTGG AGTCGCAGAG AGCCTGACCA TGTTCGACCT GACCTCAGAG CAACTGCTGG GGGCACTCAG GAAAGTCCTC AACAACAAGC GCTACAAAGA GAAGATAACA CAGCTGTCTT TGATCCATAA AGACCGTCCG ATCGAGCCGC TGGACTTGGC CGTGTTCTGG ACCGAGTTTG TGATGAGACA CGGAAGTGCC GAGCACCTGA GACCGGCCGC TCACCACCTG AACTGGATCC AGTACCAC ii. SEQUENCES OBTAINED BY 5 -RACE (1st round) >UGT_L25R (LB XI, p. 139, lane 2) AAAGCAAGAT ATTTTTCTCC AGCTTTGATG AGCTCACCAG CAGATATCTC AAGAAGGATG TTACGTTCAG AGACGTCCTC GGACATGCCG CGATTTGGCT TTATAGATAT GACTTCACCT TTGAGTACCC GAGACCTGTA ATGCCCAATG CGGTCAGAAT TGGTGGCATC AACTGTGCCA AGAAGAATCC TCTGCCTGCC GATCTGGAGG AGTTCGTGGA CGGTTCTGGA GATCACGGCT TCATCGTGTT CACTTTGGGC TCCTTCGTGT CCGAGCTGCC GGAGTTCAAA GCCCGGGAGT TTTTCGAGGC TTTTCGGCAG ATTCCTCAGA GGGTTCTGTG GCGATACACC GGGGTCATTC CCAAAGACAT TCCTGAAGAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAC GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC AC

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133 >UGT_L35R (LB XI, p. 139, lane 3) AGCTCACCAG CAGATATCTC AAGAAGGATG TTACGTTCAG AGACGTCCTC GGACATGCCG CGATTTGGCT TTATGGATAT GACTTCACCT TTGAGTACCC GAGACCTGTA ATGCCCAATG CGGTCAGAAT TGGTGGCATC AACTGTGCCA AGAAGAATCC TCTGCCTGCC GATCTGGAGG AGTTCGTGGA CGGTCCTGGA GATCACGGCT TCATCGTGTT CACTTTGGGC TCCTTCGTGT CCGAGCTGCC GGAGTTCAAA GCCCGGGAGT TTTTCGAGGC TTTTCGGCAG ATTCCTCAGA GGGTTCTGTG GCGATACACC GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAC GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAGC CCATGGCATC TACGAGGGTA TCTGT iii. SEQUENCES OBTAIN ED BY 5'-RACE (2nd round) PCR products only, not cloned >UGT_L25R_5R (LB XII, p.8, lanes 1-2; p.49, lanes 1-2) (1 additional round of amplification) GGAATGCTAA GAGCTCGAGT ACCGGGCCTG TTCTTCTCAC ATTCCTCCTC TTCTTTTTTT CCTCCAAAAT CTGCTTCCTC TAGACGTAAT TAGAAACTTT TAAGCTAAAA ATGCCTCGTC TTCTTGCAGC TCTCTGTCTC CAGATTTATC TTTGCAGCTT TTTAGGACCA GTGGAAGGAG GGAAGGTCCT GGTGATGCCC GTGGACGGCA GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC TCGGAGAGGA CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA TCCATGGCTC TGACGCGTAC GCCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGACT GGATGAAAGC ATGAATAAGT TGAAGGAGGG CATTACGAAA GCACCGCGGA TCTCTGACTT ATTGGAGAAC ATCATCGGGC TCCTCAGCTT CACGAACATG CAGGTGAAAG GATGCGAGGG CTGCTGTATA ACGAGCCTCT GATGCAGAAC CTGCGCGAGG AACACTTCGA TCTCATGCTC ACCGATCCCT TCCTGCCTTG TGGCCCCATC ATCGCCGAGG CTTTCTCCCT CCCCGCCGTT TATTTCCTGC GTGGGCTTCC CTGCGGATTG GATCTGGAAG CCGCTCAGTG CCCATCGCCT CCGTCCTACG TCCCGCGCTT TTTCACAGGC AACACCGACG TCATGACGTT TTCTCAGAGG GTCAAGAACG TGCTCATGAC GGGATTCGAG AGCATCCAAA GCAAGATATT TTTCTCCAGC TTTGATGAGC TCACCAGCAG A >UGT_L25R_5R (LB XII, p. 108, lanes 2 and 3) AATGCTAAGA GCTCGAGTAC CGGGCCTGTT CTTCTCACAT TCCTCCTCCT TCTTTTTTTC CTCCAAAATC TGCTTCCTCT AGACGTAATT AGAAACTTTT AAGCTAAAAA TGCCTCGTCT TCTTGCAGCT CTCTGTCTCC AGATTTATCT TTGCAGCTTT TTAGGACCAG TGGAAGGAGG GAAGGTCCTG GTGATGCCCG TGGACGGCAG CCACTGGCTC AGTATGAAGA TCTTGGTGGA GGAATTGTCT CGGAGAGGAC ATGAAATGGT GGTCCTGGTT CCCGAGACAA GCGTGTTGAT CCATGGCTCT GACGCGTACG CCGCTCGGAG CTTTAAGGTT CCGTACACCA AGGCTGAACT GGATGAAAGC ATGAATAAGT TGAAGGAGGG CATTACGAAA GCACCGCGGA TCTCTGACTT ATTGGAGAAC ATCATCGGGC TCCTCAGCTT CACGAACATG CAGGTGAAAG GATGCGAGGC GCTGCTGTAT AACGAGCCTC TGATGCAGAA CCTGCGCGAG GAACACTTCG ATCTCATGCT CACCGATCCC TTCCTGCCTT GTGGCCCCAT CATCGCCGAG GCTTTCTCCC TCCCCGCCGT TTATTTCCTG CGTGGGCTTC CCTGCGGATT GGATCTGGAA GCCGCTTAGT GCCCATCGCC TCCGTCCTAC GTCCCGCGCT TTTTCACAGG CAACACCGAC GTCATGACGT TTTCTCAGAG GGTCAAGAAC GTGCTCATGA CGGGATTCGA GAGCATCCTT TGCAAAATAT TTTTCTCCAG CTTTGATGAG CTCACCAGCA GA

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134 iii. SEQUENCES O BTAINED BY 3 -RACE >UGT_L25R_5A (LB XII, p. 13, lane 6) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAC GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAGC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACGAGCG CTAAAAAAAA AAA >UGT_L25R_5Av (LB XII, p. 47, lane 4) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAT GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAAC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACAAGCG CTAAAAAAAA AAAA >UGT_L25R_5Avi (LB XII, p. 47, lane 5) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAT GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAAC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACAAGCG CTACAAAGAA AAAAAAAA >UGT_L25R_4Bb (LB XII, p. 27, lane 9) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCTAAGTGA TGAGGTGGCT TCCGCAGAAC GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAGC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGGA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACAAGCG CTACAAAGAG AAGATAACAC AGCTGTCTTT GATCCATAAA GACCGTCCGA TCGAGCCGCT GGACTTGGCC GTGTTCTGGA CCGAGTTTGT GATGAGACAC GGAAGTGCCG AGCACCTGAG ACCGGCCGCT CACCACCTCA ACTGGGTTCA GTACCACAGT CTCGATGTCA TCGCCTTCCT CCTGCTCGTT CTATCCACCG TCGTTTTTAT CGCCGTCAAA ACCTGCGCGC TCTGTTTCAG GAAGTGTTTC CGGAGGGCTC AGAAGAGCAA AAAGGAGTGA AACGGCCAGT GAATGATCAG GAATGGATTT GGTGCCGTCT TTAATTAACG CCGATGGTTT ATCGGCGTGA TGTCATACTG TGAAAACCTG AAATAGTTAT AGTGTTCTCA TCACCACGTT CAATTTAATA TTCAGGGGTG CCAGCAATTA TGGTTTAGCC ATTGCAGTTA CGGTTGTTAT GATGTCACTA AAAAAAAAAA A >UGT_L25R_4Bi (LB XII, p. 47, lane 1) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAT GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAAC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACAAGCG CTACAAAGAG AAGATAACAC AGCTGTCTTT GATCCATAAA GACCGTCCGA TCGAGCCGCT GGACTTGGCC GTGTTCTGGA CCGAGTTTGT GATGAGACAC GGAAGTGCCG AGCACCTGAG ACCGGCCGCT CACCACCTCA ACTGGGTTCA GTACCACAGT CTCGATGTCA TCGCCTTCCT CCTGCTCGTT CTATCCACCG TCGTTTTTAT CGCCGTCAAA ACCTGCGCGC TCTGTTTCAG GAAGTGTTTC CGGAGGGCTC AGAAGAGCAA AAAGGAGTGA AACGGCCAGT GAATGATCAG GAATGGATTT GGTGCCGTCT TTAATTAACG CCGATGGTTT ATCGGCGTGA TGTCATACTG TGAAAACCTG AAATAGTTAT AGTGTTCTCA TCACCACGTT CAATTTAATA TTCAGGGGTG CCAGCAATTA TGGTTTAGCC ATTGCAGTTA CGGTTGTTAT GATGTCACAA AAAAAAAAAA

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135 >UGT_L25R_4Bii (LB XII, p. 47, lane 2) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAGGTGA TGAAGTGGCT TCCGCAGAAT GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAGC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACAAGCG CTACAAAGAG AAGATAACAC AGCTGTCTTT GATCCATAAA GACCGTCCGA TCGAGCCGCT GGACTTGGCC GTGTTCTGGA CCGAGTTTGT GATGAGACAC GGAAGTGCCG AGCACCTGAG ACCGGCCGCT CACCACCTCA ACTGGGTTCA GTACCACAGT CTCGATGTCA TCGCCTTCCT CCTGCTCGTT CTATCCACCG TCGTTTTTAT CGCCGTCAAA ACCTGCGCGC TCTGTTTCAG GAAGTGTTTC CGGAGGGCTC AGAAGAGCAA AAAGGAGTGA AACGGCCAGT GAATGATCAG GAATGGATTT GGTGCCGTCT TTAATTAACG CCGATGGTTT ATCGGCGTGA TGTCATACTG TGAAAACCTG AAATAGTTAT AGTGTTCTCA TCACCACGTT CAATTTAATA TTCAGGGGTG CCAGCAATTA TGGTTTAGCC ATTGCAGTTA CGGTTGTTAT GATGTCACTA AAAAAAAAAA AA >UGT_L25R_4Biii (LB XII, p. 47, lane 3) GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAT GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAAC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACAAGCG CTACAAAGAG AAGATAACAC AGCTGTCTTT GATCCATAAA GACCGTCCGA TCGAGCCGCT GGACTTGGCC GTGTTCTGGA CCGAGTTTGT GATGAGACAC GGAAGTGCCG AGCACCTGAG ACCGGCCGCT CACCACCTCA ACTGGGTTCA GTACCACAGT CTCGATGTCA TCGCCTTCCT CCTGCTCGTT CTATCCACCG TCGTTTTTAT CGCCGTCAAA ACCTGCGTGC TCTGTTTCAG GAAGTGTTTC CGGAGGGCTC AGAAGAGCAA AAAGGAGTGA AACGGCCAGT GAATGATCAG GAATGGATTT GGTGCCGTCT TTAATTAACG CCGATGGTTT ATCGGCGTGA TGTCATACTG TGAAAACCTG AAATAGTTAT AGTGTTCTCA TCACCACGTT CAATTTAATA TTCAGGGGTG CCAGCAATTA TGGTTTAGCC ATTGCAGTTA CGGTTGTTAT GATGTCACGA AAAAAAAAAA A

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136 B. 5' 3' sequences of partial length clones for intestinal UGT i. SEQUENCES OBTAINED BY DEGENERATE PRIMERS >UGT_I1 DEGENERATE (LB XI, p. 91, lane 1) GAGTTTGTGG ATGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA CGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGTG CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GTTGATGTTC CCACTGTTT >UGT_I2 DEGENERATE (LB XI, p. 91, lane 2) GAGTTTGTGA ATGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGGG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA TGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAA CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GCTGATGTTC CCACTGTTT >UGT_I3 DEGENERATE (LB XI, p. 91, lane 3) CTAGTGATTG AGTTTGTGGA TGGCTCTGGA GATCACGGCT TCATCGTGTT CACTTTGGGC TCCTTCGTGT CCGAGCTGCC GGAGTTCAAA GCCCGGGAGT TTTTCGAGGC TTTTCGGCAG ATTCCTCAGA GGGTTCTGTG GCGATACACC GGGGTCATTC CCAAAGACAT TCCTGAAAAT GTCAAAGTGA TGAAGTGGCT TCCGCAGAAC GACCTCTTAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAGC CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG TTGATGTTCC CTCTGTTT >UGT_I4 DEGENERATE (LB XI, p. 91, lane 4) GAGTTTGTGG AAGGCTCAGG AGATCACGGC TTCATCGTGT TCACTTTGAG CTCCTTCGTG TCCGAGCTGC CGGAGTTCAA AGCCCGGGAG TTTTTCGAGG CTTTTCGGCA GATTCCTCAG AGGGTTCTGT GGCGATACAC CGGGGTCATT CCCAAAGACA TTCCTGAAAA TGTCAAAGTG ATGAAGTGGC TTCCGCAGAA CGACCTCTTA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGTG CCCATGGCAT CTACGAGGGT ATCTGTAACG GCGTGCCGAT GTTGATGTTC CCACTGTTT ii. SEQUENCES OBTAINED BY 5 -RACE >UGT_I15R (LB XI, p. 139, lane 5) AAATTCCCAA GGACATTCCT GAAAATGTCA AAGTGATGAA GTGGCTTCCG CAGAATGACC TCTTAGGTTT GTTTACACGT CCTCTAACCG TAATAAATAG ACACCCGGTC CCCATTTCTC TCACACACAC ACACATCTAT CTATCACGCA GGTCTATGAT TATCGATTAT ACCGTACGTT TCCAGCTAAC ACTACTTGGA TACTTTGGTC AAAAACTCAC ACCGAAGGTC ATTAACACAC AGTTCCTGTT TTAAACAGCG TTAAAATTTA AATCTGAAAG ATTCGAGGAA ATATAATGGT GCATAATAAT AATTTCCTTT TTTCTTTCCT TTCATCGCCG TGTTAAAAAG CACACCCCAA GGCTAAGGTG TTCATCACGC ACGGAGGAAC CCATGGCATC TACGAGGGTA TCTGT >UGT_I25R (LB XI, p. 139, lane 6) AAATTCCCAA AGACATTCCT GAAAATGTCA AAGTGATGAA GTGGCTTCCG CAGAATGACC TCTTAGGTTT GTTTACACGT CCTCTAACCG TAATAAATAG ACACCCGGTC CCCATTTTCT CTCACACACA CACACATCTA TCTATCACAC AGGTCTATGA TTATCGATTA TACCGTACGT TTCCAGCTAA CACTACTTGG ATACTTTGGT CAAAAACTCA CACCGAAGGT CATTAACACA CAGTTCCTGT TTTAAACAGC GTTAAAATTT AAATCTGAAA GATTCGAGGA AATATAATGG TGCATAATAA TAATTTCCTT TTTTCTTTCC TTTCATCGCC GTGTTAAAAA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAA CCCATGGCAT CTACGAGGGT ATCTGT

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137 >UGT_I35R (LB XI, p. 139, lane 7) AAATTCCCAA AGACATTCCT GAAAATGTCA AAGTGATGAA GTGGCTTCCG CAGAATGACC TCTTAGGTTT GTTTACACGT CCTCTAACCG TAATAAATAG ACACCCGGTC CCCATTTTCT CTCTCACACA CACACATCTA TCTATCACAC AGGTCTATGA TTATCGATTA TACCGTACGT TTCCAGCTAA CACTACTTGG ATACTTTGGT CAAAAACTCA CACCGAAGGT CATTAACACA CAGTTCCTGT TTTAAACAGC GTTAAAATTT AAATCTGAAA GATTCGAGGA AATATAATGG TGCATAATAA TAATTTCCTT TTTTCTTTCC TTTCATCGCC GTGTTAAAAA GCACACCCCA AGGCTAAGGT GTTCATCACG CACGGAGGAA CCCATGGCAT CTACGAGGGT ATCTGT iii. SEQUENCES OBTA INED BY 3'-RACE >UGT_I4_6A (LB XII, p. 27, lane 5) CCCAAGGCTA AGGTGTTCAT CACGCACGGA GGAGCCCATG GCATCTACGA GGGTATCTGT AACGGCGTGC CGATGGTGAT GATCCCGCTG TTCGGAGATC AGGTAGACAA CGTTCTACGC ATGGTGCTGC GTGAAGTCGC AGAGAGCCTG ACCATGTTCG ACCTGACCTC AGAGCAACTG CTGGGGGCAC TCAGGAAAGT CCTCAACAAC GAGCGCCAAA AAAAAAAAAA >UGT_I46Aix (LB XII, p. 47, lane 6) CCCAAGGCTA AGGTGTTCAT CACGCACGGA GGAGCCCATG GCATCTACGA GGGTATCTGT AACGGCGTGC CGATGGTGAT GATCCCGCTG TTCGGAGATC AGGTAGACAA CGTTCTACGC ATGGTGCTGC GTGAAGTCGC AGAGAGCCTG ACCATGTTCG ACCTGACCTC AGAGCAACTG CTGGGGGCAC TCAGGAAAGT CCTCAACAAC GAGCGCTAAA AAAAA >UGT_I46Ax (LB XII, p. 47, lane 7) CCCAAGGCTA AGGTGTTCAT CACGCACGGA GGAGCCCATG GCATCTACGA GGGTATCTGT AACGGCGTGC CGATGGTGAT GATCCCGCTG TTCGGAGATC AGGTAGACAA CGTTCTACGC ATGGTGCTGC GTGAAGTCGC AGAGAGCCTG ACCATGTTCG ACCTGACCTC AGAGCAACTG CTGGGGGCAC TCAGGAAAGT CCTCAACAAC GAGCGCTAAA AAAAAAAA The following sequences were all sequenced directly from the PCR product and were not cloned >I4_3R (LB XII, p.128, lower lanes 4-5; p.142, lane 1) CACGCACGGA GGAACCCATG GCATCTACGA GGGTATCTGT AACGGCGTGC CGATGGTGAT GATCCCGCTG TTCGGAGATC AGGTAGACAA CGTTCTACGC ATGGTGCTGC GTGAAGTCGC AGAGAGCCTG ACCATGTTCG ACCTGACCTC AGAGCAACTG CTGGGGGCAC TCAGGAAAGT CCTCAACAAC AAGCGCTACA AAGAGAAGAT AACACAGCTG TCTTTGATCC ATAAAGACCG TCCGATCGAG CCGCTGGACT TGGCCGTGTT CTGGACCGAG TTTGTGATGA GACACGGAAG TGCCGAGCAC CTGAGACCGG CCGCTCACCA CCTCAACTGG GTTCAGTACC ACAGTCTCGA TGTCATCGCC TTCCTCCTGC TCGTTCTATC CACCGTCGTT TTTATCGCCG TCAAAACCTG CGTGCTCTGT TTCAGGAAGT GTTTCCGGAG GGCTCAGAAG AGCAAAAAGG AGTGAAACGG CCAGTGAATG ATCAGGAATG GATTTGGTGC CGTCTTTAAT TAACGCCGAT GGTTTATCGG CGTGATGTCA TACTGTGAAA ACCTGAAATA GTTATAGTGT TCTCATCACC ACGTTCAATT TAATATTCAG GGGTGCCAGC AATTATGGTT TAGCCATTGC AGTTACGGTT GTTATGATGT CACGAAAAAA AAAAA >I35R_PCR (LB XII, p.126, band 6, lane 6) AGCCCATGGC ATCTACGAGG GTATCTGTAA CGGCGTGCCG ATGGTGATGA TCCCGCTGTT CGGAGATCAG GTAGACAACG TTCTACGCAT GGTGCTGCGT GAAGTCGCAG AGAGCCTGAC CATGTTCGAC CTGACCTCAG AGCAACTGCT GGGGGCACTC AGGAAAGTCC TCAACAACGA GCGCTAAAAA AAAAA >I35R_PCR2 (LB XII, p.126, band 6A, lane 6; p.130, lanes 3-4) CCATGGCATC TACGAGGGTA TCTGTAACGG CGTGCCGATG GTGATGATCC CGCTGTTCGG AGATCAGGTA GACAACGTTC TACGCATGGT GCTGCGTGAA GTCGCAGAGA GCCTGACCAT GTTCGACCTG ACCTCAGAGC AACTGCTGGG GGCACTCAGG AAAGTCCTCA ACAACGAGCG CTAAAAAAAA AA

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138 APPENDIX B SEQUENCES FOR UGT FULL-LENGTH CLONES FROM CATFISH LIVER A. UGT clones with UTRs at either end (h ighlighted area indicates start and stop codons) >UTR1 1 CTGCTTCCTC TAGACGTAAT TAGAAACTTT TAAGCTAAAA ATGCTTCGTC TTCTTGCAGC 61 TCTCTGTCTC CAGATTTATC TTTGCAGCTT TTTAGGACCA GTGGAAGGAG GGAAGGTCCT 121 GGTGATGCCC GTGGACGGCA GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC 181 TCGGAGAGGA CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA TCCATGGCTC 241 TGACGCGTAC GCCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGAAC TGGATGAAAG 301 CATGAATAAG TTGAAGGAGG GCATTACGAA AGCACCGCGG ATCTCTGACT TATTGGAGAA 361 CATCATCGGG CTCCTCAGCT TCACGAACAT GCAGGTGAAA GGATGCGAGG CGCTGCTGTA 421 TAACGAGCCT CTGATGCAGA ACCTGCGCGA GGAACACTTC GATCTCATGC TCACCGATCC 481 CTTCCTGCCT TGTGGCCCCA TCATCGCCGA GGCTTTCTCC CTCCCCGCCG TTTATTTCCT 541 GCGTGGGCTT CCCTGCGGAT TGGATCTGGA AGCCACTCAG TGCCCATCGC CTCCGTCCTA 601 CGTCCCGCGC TTTTTCACAG GCAACACCGA CGTCATGACG TTTTCTCAGA GGGTCAAGAA 661 CGTGCTCATG ACGGGATTCG AGAGCATCCT TTGCAAAATA TTTTTCTCCA GCTTTGATGA 721 GCTCACCAGC AGATATCTCA AGAAGGATGT TACGTTCAGA GACGTCCTCG GACATGCCGC 781 GATTTGGCTT TATAGATATG ACTTCACCTT TGAGTACCCG AGACCTGTAA TGCCCAATGC 841 GGTCAGAATT GGTGGCATCA ACTGTGCCAA GAAGAATCCT CTGCCTGCCG ATCTGGAGGA 901 GTTCGTGGAC GGTTCTGGAG ATCACGGCTT CATCGTGTTC ACTTTGGGCT CCTTCGTGTC 961 CGAGCTGCCG GAGTTCAAAG CCCGGGAGTT TTTCGAGGCT TTTCGGCAGA TTCCTCAGAG 1021 GGTTCTGTGG CGATACACCG GGGTCATTCC CAAAGACATT CCTGAAAATG TCAAAGTGAT 1081 GAAGTGGCTT CCGCAGAACG ACCTCTTAGC ACACCCAAGG CTAAGGTGTT CATCACGCAC 1141 GGAGGAGCCC ATGGCATCTA CGAGGGTATC TGTAACGGCG TGCCGATGGT GATGATCCCG 1201 CTGTTCGGAG ATCAGGTAGA CAGCGTTCTA CGCATGGTGC TGCGTGGAGT CGCAGAGAGC 1261 CTGACCATGT TCGACCTGAC CTCAGAGCAA CTGCTGGGGG CACTCAGGAA AGTCCTCAAC 1321 AACAAGCGCT ACAAAGAGAA GATAACACAG CTGTCTTTGA TCCATAAAGA CCGTCCGATC 1381 GAGCCGCTGG ACTTGGCCGT GTTCTGGACC GAGTTTGTGA TGAGACACGG AAGTGCCGAG 1441 CACCTGAGAC CGGCCGCTCA CCACCTCAAC TGGGTTCAGT ACCACAGTCT CGATGTCATC 1501 GCCTTCCTCC TGCTCGTTCT ATCCACCGTC GTTTTTATCG CCGTCAAAAC CTGCGCGCTC 1561 TGTTTCAGGA AGTGTTTCCG GAGGGCTCAG AAGAGCAAAA AGGAGTGAAA CGGCCAGTGA 1621 ATGATCAGGA ATGGATTTGG TGCCGTCTTT AATTAACGCC GATGGTTTAT CGGCGTGATG 1681 TCATACTGTG AAAACCTGAA ATAGTTATAG TGTTCTCATC ACCACGTTCA ATTTAATATT 1741 CAGGGGTGCC AGCAATTATG GTTTAGCCAT TGCAGTTACG GT

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139 >UTR2 1 CTGCTTCCTC TAGACGTAAT TAGAAACTTT TAAGCTAAAA ATGCCTCGTC TTCTTGCAGC 61 TCTCTGTCTC CAGATTTATC TTTGCAGCTT TTTAGGACCA GTGGAAGGAG GGAAGGTCCT 121 GGTGATGCCC GAGGACGGCA GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC 181 TCGGAGAGGA CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA TCCATGGCTC 241 TGACGCGTAC GTCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGAAC TGGATGAAAG 301 CATGAATAAG TTGAAGGAGG GCATTACGAA AGCACCGCGG ATCTCTGACT TATTGGAGAA 361 CATCATCGGG CTCCTCAGCT TCACGAACAT GCAGGTGAAA GGATGCGAGG CGCTGCTGTA 421 TAACGAGCCT CTGATGCAGA ACCTGCGCGA GGAACACTTC GATCTCATGC TCACCGATCC 481 CTTCCTGCCT TGTGGCCCCA TCATCGCCGA GGCTTTCTCC CTCCCCGCCG TTTATTTCCT 541 GCGTGGGCTT CCCTGCGGAT TGGATCTGGA AGCCGCTCAG TGCCCATCGC CTCCGTCCTA 601 CGTCCCGCGC TTTTTCACAG GCAACACCGA CGTCATGACG TTTTCTCAGA GGGTCAAGAA 661 CGTGCTCATG ACGGGATTCG AGAGCATCCT TTGCAAAATA TTTTTCTCCA GCTTTGATGA 721 GCTCACCAGC AGATATCTCA AGAAGGATGT TACGTTCAGA GACGTCCTCG GACATGCCGC 781 AATTTGGCTT TATAGATATG ACTTCACCTT TGAGTACCCG AGACCTGTAA TGCCCAATGC 841 GGTCAGAATT GGTGGCATCA ACTGTGCCAA GAAGAATCCT CTGCCTGCCG ATCTGGAGGA 901 GTTCGTGGAC GGTTCTGGAG ATCACGGCTT CATCGTGTTC ACTTTGGGCT CCTTCGTGTC 961 CGAGCTGCCG GAGTTCAAAG CCCGGGAGTT TTTCGAGGCT TTTCGGCAGA TTCCTCAGAG 1021 GGTTCTGTGG CGATACACCG GGGTCATTCC CAAAGACATT CCTGAAAATG TCAAAGTGAT 1081 GAAGTGGCTT CCGCAGAATG TCCTCTTAGC ACACCCCAAG GCTAAGGTGT TCATCACGCA 1141 CGGAGGAACC CATGGCATCT ACGAGGGTAT CTGTAACGGC GTGCCGATGG TGATGATCCC 1201 GCTGTTCGGA GATCAGGTAG ACAACGTTCT ACGCATGGTG CTGCGTGAAG TCGCAGAGAG 1261 CCTGACCATG TTCGACCTGA CCTCAGAGCA ACTGCTGGGG GCACTCAGGA AAGTCCTCAA 1321 CAACAAGCGC TACAAAGAGA GGATAACACA GCTGTCTTTG ATCCATAAAG ACCGTCCGAT 1381 CGAGCCGCTG GACTTGGCCG TGTTCTGGAC CGAGTTTGTG ATGAGACACG GAAGTGCCGA 1441 GCACCTGAGA CCGGCCGCTC ACCACCTCAA CTGGGTTCAG TACCACAGTC TCGATGTCAT 1501 CGCCTTCCTC CTGCTCGTTC TATCCACCGT CGTTTTTATC GCCGTCAAAA CCTGCGCGCT 1561 CTGTTTCAGG AAGTGTTTCC GGAGGGCTCA GAAGAGCAAA AAAGAGTGAA ACGGCCAGTG 1621 AATGATCAGG AATGGATTTG GTGCCGTCTT TAATTAACGC CGATGGTTTA TCGGCGTGAT 1681 GTCATACTGT GAAAACCTGA AATAGTTATA GTGTTCTCAT CACCACGTTC AATTTAATAT 1741 TCAGGGGTGC CAGCAATTAT GGTTTAGCCA TTGCAGTTAC GGTTGTTATG ATGTCACTA

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140 >UTR3 1 CTGCTTCCTC TAGACGTAAT TAGAAACTTT TAAGCTAAAA ATGCCTCGTC TTCTTGCAGC 61 TCTCTGTCTC CAGATTTATC TTTGCAGCTT TTTAGGACCA GTGGAAGGAG GGAAGGTCCT 121 GGTGATGCCC GTGGACGGCA GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC 181 TCGGAGAGGA CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA TCCATGGCTC 241 TGACGCGTAC GTCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGAAC TGGATGAAAG 301 CATGAATAAG TTGAAGGAGG GCATTACGAA AGCACCGCGG ATCTCTGACT TATTGGAGAA 361 CATCATCGGG CTCCTCAGCT TCACGAACAT GCAGGTGAAA GGATGCGAGG CGCTGCTGTA 421 TAACGAGCCT CTGATGCAGA ACCTGCGCGA GGAACACTTC GATCTCATGC TCACCGATCC 481 CTTCCTGCCT TGTGGCCCCA TCATCGCCGA GGCTTTCTCC CTCCCCGCCG TTTATTTCCT 541 GCGTGGGCTT CCCTGCGGAT TGGATCTGGA AGCCGCTCAG TGCCCATCGC CTCCGTCCTA 601 CGTCCCGCGC TTTTTCACAG GCAACACCGA CGTCATGACG TTTTCTCAGA GGGTCAAGAA 661 CGTGCTCATG ACGGGATTCG AGAGCATCCT TTGCAAAATA TTTTTCTCCA GCTTTGATGA 721 GCTCACCAGC AGATATCTCA AGAAGGATGT TACGTTCAGA GACGTCCTCG GACATGCCGC 781 AATTTGGCTT TATAGATATG ACTTCACCTT TGAGTACCCG AGACCTGTAA TGCCCAATGC 841 GGTCAGAATT GGTGGCATCA ACTGTGCCAA GAAGAATCCT CTGCCTGCCG ATCTGGAGGA 901 GTTCGTGGAC GGTTCTGGAG ATCACGGCTT CATCGTGTTC ACTTTGGCTC CTTCGTGTCC 961 GAGCTGCCGG AGTTCAAAGC CCGGGAGTTT TTCGAGGCTT TTCGGCAGAT TCCTCAGAGG 1021 GTTCTGTGGC GATACACCGG GGTCATTCCC AAAGACATTC CTGAAAATGT CAAAGTGATG 1081 AAGTGGCTTC CGCAGAATGA CCTCTTAGCA CACCCCAAGG CTAAGGTGTT CATCACGCAC 1141 GGAGGAACCC ATGGCATCTA CGAGGGTATC TGTAACGGCG TGCCGATGGT GATGATCCCG 1201 CTGTTCGGAG ATCAGGTAGA CAACGTTCTA CGCATGGTGC TGCGTGAAGT CGCAGAGAGC 1261 CTGACCATGT TCGACCTGAC CTCAGAGCAA CTGCTGGGGG CACTCAGGAA AGTCCTCAAC 1321 AACAAGCGCT ACAAAGAGAA GATAACACAG CTGTCTTTGA TCCATAAAGA CCGTCCGATC 1381 GAGCCGCTGG ACTTGGCCGT GTTCTGGACC GAGTTTGTGA TGAGACACGG AAGTGCCGAG 1441 CACCTGAGAC CGGCCGCTCA CCACCTCAAC TGGGTTCAGT ACCACAGTCT CGATGTCATC 1501 GCCTTCCTCC TGCTCGTTCT ATCCACCGTC GTTTTTATCG CCGTCAAAAC CTGCGCGCTC 1561 TGTTTCAGGA AGTGTTTCCG GAGGGCTCAG AAGAGCAAAA AGGAGTGAAA CGGCCAGTGA 1621 ATGATCAGGA ATGGATTTGG TGCCGTCTTT AATTAACGCC GATGGTTTAT CGGCGTGATG 1681 TCATACTGTG AAAACCTGAA ATAGTTATAG TGTTCTCATC ACCACGTTCA ATTTAATATT 1741 CAGGGGTGCC AGCAATTATG GTTTAGCCAT TGCAGTTACG GTTGTTATGA TGTCACTA As explained in Chapter 5, the full-length intestinal UGT clones were identical in sequence to the UTR sequences shown above for liver.

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141 B. UGT clones comprising tr anslated portion of gene >UGT1 1 ATGCCTCGTC TTCTTGCAGC TCTCTGTCTC CAGATTTATC TTTGCAGCTT 51 TTTAGGACCA GTGGAAGGAG GGAAGGTCCT GGTGATGCCC GTGGACGGCA 101 GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC TCGGAGAGGA 151 CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA TCCATGGCTC 201 TGACGCGTAC GTCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGAAC 251 TGGATGAAAG CATGAATAAG TTGAAGGAGG GCATTACGAA AGCACCGCGG 301 ATCTCTGACT TATTGGAGAA CATCATCGGG CTCCTCAGCT TCACGAACAT 351 GCAGGTGAAA GGATGCGAGG CGCTGCTGTA TAACGAGCCT CTGATGCAGA 401 ACCTGCGCGA GGAACACTTC GATCTCATGC TCACCGATCC CTTCCTGCCT 451 TGTGGCCCCA TCATCGCCGA GGCTTTCTCC CTCCCCGCCG TTTATTTCCT 501 GCGTGGGCTT CCCTGCGGAT TGGATCTGGA AGCCGCTCAG TGCCCATCGC 551 CTCCGTCCTA CGTCCCGCGC TTTTTCACAG GCAACACCGA CGTCATGACG 601 TTTTCTCAGA GGGTCAAGAA CGTGCTCATG ACGGGATTCG AGAGCATCCT 651 TTGCAAAATA TTTTTCTCCA GCTTTGATGA GCTCACCAGC AGATATCTCA 701 AGAAGGATGT TACGTTCAGA GACGTCCTCG GACATGCCGC AATTTGGCTT 751 TATAGATATG GCTTCACCTT TGAGTACCCG AGACCTGTAA TGCCCAATGC 801 GGTCAGAATT GGTGGCATCA ACTGTGCCAA GAAGAATCCT CTGCCTGCCG 851 ATCTGGAGGA GTTCGTGGAC GGTTCTGGAG ATCACGGCTT CATCGTGTTC 901 ACTTTGGGCT CCTTCGTGTC CGAGCTGCCG GAGTTCAAAG CCCGGGAGTT 951 TTTCGAGGCT TTTCGGCAGA TTCCTCAGAG GGTTCTGTGG CGATACACCG 1001 GGGTCATTCC CAAAGACATT CCTGAAAATG TCAAAGTGAT GAAGTGGCTT 1051 CCGCAGAATG ACCTCTTAGC ACACCCCAAG GCTAAGGTGT TCATCACGCA 1101 CGGAGGAACC CATGGCATCT ACGAGGGTAT CTGTAACGGC GTGCCGATGG 1151 TGATGATCCC GCTGTTCGGA GATCAGGTAG ACAACGTTCT ACGCATGGTG 1201 CTGCGTGAAG TCGCAGAGAG CCTGACCATG TTCGACCTGA CCTCAGAGCA 1251 ACTGCTGGGG GCACTCAGGA AAGTCCTCAA CAACAAGCGC TACAAAGAGA 1301 AGATAACACA GCTGTCTTTG ATCCATAAAG ACCGTCCGAT CGAGCCGCTG 1351 GACTTGGCCG TGTTCTGGAC CGAGTTTGTG ATGAGACACG GAAGTGCCGA 1401 GCACCTGAGA CCGGCCGCTC ACCACCTCAA CTGGGTTCAG TACCACAGTC 1451 TCGATGTCAT CGCCTTCCTC CTGCTCGTTC TATCCACCGT CGTTTTTATC 1501 GCCGTCAAAA CCTGCGCGCT CTGTTTCAGG AAGTGTTTCC GGAGGGCTCA 1551 GAAGAGCAAA AAGGAGTGA

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142 >UGT2 1 ATGCCTCGTC TTCTTGCAGC TCTCTGTCTC CAGATTTATC TTTGCAGCTT 51 TTTAGGACCA GTGGAAGGAG GGAAGGTCCT GGTGATGCCC GTGGACGGCA 101 GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC TCGGAGAGGA 151 CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA CCCATGGCTC 201 TGACGCGTAC GTCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGAAC 251 TGGATGAAAG CATGAATAAG TTGAAGGAGG GCATTACGAA GGCACCGCGG 301 ATCTCTGACT TATTGGAGAA CATCATCGGG CTCCTCAGCT TCACGAACAT 351 GCAGGTGAAA GGATGCGAGG CGCTGCTGTA TAACGAGCCT CTGATGCAGA 401 ACCTGCGCGA GGAACACTTC GATCTCATGC TCACCGATCC CTTCCTGCCT 451 TGTGGCCCCA TCATCGCCGA GGCTTTCTCC CTCCCCGCCG TTTATTTCCT 501 GCGTGGGCCT CCCTGCGGAT TGGATCTGGA AGCCGCTCAG TGCCCATCGC 551 CTCCGTCCTA CGTCCCGCGC TTTTTCACAG GCAACACCGA CGTCATGACG 601 TTTTCTCAGA GGGTCAAGAA CGTGCTCATG ACGGGATTCG AGAGCATCCT 651 TTGCAAAATA TTTTTCTCCA GCTTTGATGA GCTCACCAGC AGATATCTCA 701 AGAAGGATGT TACGTTCAGA GACGTCCTCG GACATGCCGC AATTTGGCTT 751 TATAGATATG ACTTCACCTT TGAGTACCCG AGACCTGTAA TGCCCAATGC 801 GGTCAGAATT GGTGGCATCA ACTGTGCCAA GAAGAATCCT CTGCCTGCCG 851 ATCTGGAGGA GTTCGTGGAC GGTTCTGGAG ATCACGGCTT CATCGTGTTC 901 ACTTTGGGCT CCTTCGTGTC CGAGCTGCCG GAGTTCAAAG CCCGGGAGTT 951 TTTCGAGGCT TTTCGGCAGA TTCCTCAGAG GGTTCTGTGG CGATACACCG 1001 GGGTCATTCC CAAAGACATT CCTGAAAATG TCAAAGTGAT GAAGTGGCTT 1051 CCGCAGAATG ACCTCTTAGC ACACCCCAAG GCTAAGGTGT TCATCACGCA 1101 CGGAGGAACC CATGGCATCT ACGAGGGTAT CTGTAACGGC GTGCCGATGG 1151 TGATGATCCC GCTGTTCGGA GATCAGGTAG ACAACGTTCT ACGCATGGTG 1201 CTGCGTGAAG TCGCAGAGAG CCTGACCATG TTCGACCTGA CCTCAGAGCA 1251 ACTGCTGGGG GCACTCAGGA AAGTCCTCAA CAACAAGCGC TACAAAGAGA 1301 AGATAACACA GCTGTCTTTG ATCCATAAAG ACCGTCCGAT CGAGCCGCTG 1351 GACTTGGCCG TGTTCTGGAC CGAGTTTGTG ATGAGACACG GAAGTGCCGA 1401 GCACCTGAGA CCGGCCGCTC ACCACCTCAA CTGGGTTCAG TACCACAGTC 1451 TCGATGTCAT CGCCTTCCTC CTGCTCGTTC TATCCACCGT CGTTTTTATC 1501 GCCGTCAAAA CCTGCGCGCT CTGTTTCAGG AAGTGTTTCC GGAGGGCTCA 1551 GAAGAGCAAA AAGGAGTGA

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143 >UGT3 1 ATGCCTCGTC TTCTTGCAGC TCTCTGTCTC CAGATTTATC TTTGCAGCTT 51 TTTAGGACCA GTGGAAGGAG GGAAGGTCCT GGTGATGCCC GTGGACGGCA 101 GCCACTGGCT CAGTATGAAG ATCTTGGTGG AGGAATTGTC TCGGAGAGGA 151 CATGAAATGG TGGTCCTGGT TCCCGAGACA AGCGTGTTGA TCCATGGCTC 201 TGACGCGTAC GCCGCTCGGA GCTTTAAGGT TCCGTACACC AAGGCTGAAC 251 TGGATGAAAG CATGAATAAG TTGAAGGAGG GCATTACGAA AGCACCGCGG 301 ATCTCTGACT TATTGGAGAA CATCATCGGG CTCCTCAGCT TCACGAACAT 351 GCAGGTGAAA GGATGCGAGG CGCTGCTGTA TAACGAGCCT CTGATGCAGA 401 ACCTGCGCGA GGAACACTTC GATCTCATGC TCACCGATCC CTTCCTGCCT 451 TGTGGCCCCA TCATCGCCGA GGCTTTCTCC CTCCCCGCCG TTTATTTCCT 501 GCGTGGGCTT CCCTGCGGAT TGGATCTGGA AGCCACTCAG TGCCCATCGC 551 CTCCGTCCTA CGTCCCACGC TTTTTCACAG GCAACACCGA CGTCATGACG 601 TTTTCTCAGA GGGTCAAGAA CGTGCTCATG ACGGGATTCG AGAGCATCCT 651 TTGCAAAATA TTTTTCTCCA GCTTTGATGA GCTCACCAGC AGATATCTCA 701 AGAAGGATGT TACGTTCAGA GACGTCCTCG GACATGCCGC GATTTGGCTT 751 TATAGATATG ACTTCACCTT TGAGTACCCG AGACCTGTAA TGCCCAATGC 801 GGTCAGAATT GGTGGCATCA ACTGTGCCAA GAAGAATCCT CTGCCTGCCG 851 ATCTGGAGGA GTTCGTGGAC GGTTCTGGAG ATCACGGCTT CATCGTGTTC 901 ACTTTGGGCT CCTTCGTGTC CGAGCTGCCG GAGTTCAAAG CCCGGGAGTT 951 TTTCGAGGCT TTTCGGCAGA TTCCTCAGAG GGTTCTGTGG CGATACACCG 1001 GGGTCATTCC CAAAGACATT CCTGAAAATG TCAAAGTGAT GAAGTGGCTT 1051 CCGCAGAACG ACCTCTTAGC ACACCCCAAG GCTAAGGTGT TCATCACGCA 1101 CGGAGGAGCC CATGGCATCT ACGAGGGTAT CTGTAACGGC GTGCCGATGG 1151 TGATGATCCC GCTGTTCGGA GATCAGGTAG ACAACGTTCT ACGCATGGTG 1201 CTGCGTGGAG TCGCAGAGAG CCTGACCATG TTCGACCTGA CCTCAGAGCA 1251 ACTGCTGGGG GCACTCAGGA AAGTCCTCAA CAACAAGCGC TACAAAGAGA 1301 AGATAACACA GCTGTCTTTG ATCCATAAAG ACCGTCCGAT CGAGCCGCTG 1351 GACTTGGCCG TGTTCTGGAC CGAGTTTGTG ATGAGACACG GAAGTGCCGA 1401 GCACCTGAGA CCGGCCGCTC ACCACCTCAA CTGGGTTCAG TACCACAGTC 1451 TCGATGTCAT CGCCTTCCTC CTGCTCGTTC TATCCACCGT CGTTTTTATC 1501 GCCGTCAAAA CCTGCGCGCT CTGTTTCAGG AAGTGTTTCC GGAGGGCTCA 1551 GAAGAGCAAA AAGGAGTGA

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157 BIOGRAPHICAL SKETCH James Sacco was born on October 30th, 1971, in the small island nation of Malta in the middle of the Mediterranean Sea. He grad uated as a pharmacist from the University of Malta in 1992 and obtained a M.Phil. in pharmaceutical science from the same institution in 1998. While in Malta he was a volunteer in several environmental groups acting as youth secretary, public relations offi cer and newsletter editor for Nature Trust (Malta). From 2001 to 2006 he enrolled in the Ph.D. program at the Department of Medicinal Chemistry at the University of Florida where he conduc ted research on the enzymological, analytical, and molecular biolog ical aspects of the biotransformation of xenobiotics by organisms such as polar bears an d channel catfish. During this period he presented his work at several international conferences and has publ ished several papers on the topic. He was a three-time finalist in the College of Pharmacy Annual Research Showcase, and the recipient of an Intern ational Student Award in 2006. He currently resides in Madison, Wiscons in, with his two cats.